CN116964197A

CN116964197A - Novel engineered and chimeric nucleases

Info

Publication number: CN116964197A
Application number: CN202280020679.3A
Authority: CN
Inventors: 布莱恩·C·托马斯; 克利斯多佛·布朗; 克里斯蒂娜·布特弗尔德; 林俊良; 亚伦·布鲁克斯; 莫拉伊玛·M·特莫彻-迪亚兹; 格雷格·科斯特; 丽贝卡·拉莫特
Original assignee: Macrogenomics
Current assignee: Macrogenomics
Priority date: 2021-01-22
Filing date: 2022-01-21
Publication date: 2023-10-27

Abstract

Disclosed herein are engineered nucleases and nuclease systems comprising chimeric nucleases and chimeric nuclease systems. The engineered and chimeric nucleases disclosed herein comprise nucleic acid guided nucleases. Further disclosed herein are methods of producing the engineered nucleases and methods of using the engineered nucleases.

Description

Novel engineered and chimeric nucleases

Cross reference to related applications

The present application relates to International application No. PCT/US2021/031136 entitled "enzyme with RUVC domain (ENZYMES WITH RUVC DOMAINS)" filed on month 5 and 6 of 2021 and PCT/US2020/018432 entitled "enzyme with RUVC domain (ENZYMES WITH RUVC DOMAINS)" filed on month 2 and 14 of 2020, each of which is incorporated herein by reference in its entirety.

The present application claims the benefit of U.S. provisional application No. 63/237,484 entitled "NOVEL engineered and chimeric NUCLEASES (NOVEL ENGINEERED AND CHIMERIC NUCLEASES)" filed on month 26 of 2021 and U.S. provisional application No. 63/140,620 entitled "NOVEL engineered and chimeric NUCLEASES (NOVEL ENGINEERED AND CHIMERIC NUCLEASES)" filed on month 22 of 2021, each of which is incorporated herein by reference in its entirety.

Background

Cas enzymes and their associated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (RNAs) appear to be a common component of the prokaryotic immune system (about 45% bacteria, about 84% archaebacteria) for protecting such microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, by CRISPR-RNA-guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) element encoding the CRISPR RNA element may be relatively conserved in structure and length, its CRISPR-associated (Cas) protein is highly diverse, containing a variety of nucleic acid interaction domains. While CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capability of CRISPR/Cas complexes was not until recently recognized, leading to the use of recombinant CRISPR/Cas systems in a variety of DNA manipulation and gene editing applications.

Disclosure of Invention

In some aspects, the present disclosure provides a fusion endonuclease comprising: (a) An N-terminal sequence comprising at least a portion of a RuvC domain, REC domain, or HNH domain of an endonuclease or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO 696; and (b) a C-terminal sequence comprising a WED, TOPO or CTD domain of an endonuclease or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOS: 697-721, wherein said N-terminal sequence and said C-terminal sequence are not naturally present together in the same reading frame. In some embodiments, the endonuclease is a type II, type II Cas endonuclease. In some embodiments, the endonuclease is a class II, V Cas endonuclease. In some embodiments, the N-terminal sequence and the C-terminal sequence are derived from different organisms. In some embodiments, the N-terminal sequence further comprises a RuvC-I, BH or RuvC-II domain. In some embodiments, the C-terminal sequence further comprises a PAM interaction domain. In some embodiments, the fusion endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS. In some embodiments, the fusion endonuclease is configured to bind to PAM (SEQ ID NO: 53) that is not nnRGGnT. In some embodiments, the fusion endonuclease is configured to bind to PAM comprising any of SEQ ID NOS: 46-52 or 54-66.

In some aspects, the disclosure provides an endonuclease comprising an engineered amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs 1-27 or 108, or a variant thereof.

In some aspects, the disclosure provides an endonuclease comprising an engineered amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs 109-110, or a variant thereof.

In some aspects, the present disclosure provides a nucleic acid comprising a sequence encoding any one of the endonucleases, fusion endonucleases, or Cas enzymes described herein. In some aspects, the sequence is codon optimized for expression in a host cell. In some embodiments, the host cell is prokaryotic, eukaryotic, mammalian, or human.

In some aspects, the present disclosure provides a vector comprising any of the nucleic acid sequences described herein.

In some aspects, the present disclosure provides a host cell comprising any one of the vectors, systems, or nucleic acids described herein. In some embodiments, the host cell is prokaryotic, eukaryotic, mammalian, or human.

In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) Any of the nucleases, cas enzymes or fusion endonucleases described herein; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence; wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the endonuclease is derived from an uncultured microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12 c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the system further comprises Mg ²⁺ Is a source of (a). In some embodiments, the endonuclease comprises any of SEQ ID NOs 8-12, 26-27 or 108What has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or a variant thereof. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a non-degenerate nucleotide of any one of SEQ ID NOs 33, 34, 44, 45, 78, 84 or 87.

In some aspects, the present disclosure provides an engineered nuclease comprising: (a) A class II, type II Cas enzyme RuvC and HNH domain, or a variant thereof, having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the RuvC and HNH domain of any of SEQ ID NOs 1-27, 108 or 109-110; and (b) a class II, type II Cas enzyme PAM Interaction (PI) domain or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the PAM Interaction (PI) domain of any one of SEQ ID NOs 1-27, 108, or 109-110. In some embodiments, (a) and (b) do not naturally occur together. In some embodiments, the class II, type II Cas enzyme is derived from an uncultured microorganism. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the engineered nuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs 1-27, or a variant thereof.

In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) Any of the endonucleases described herein; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a non-degenerate nucleotide of any one of SEQ ID NOS 28-32 or 33-44, or a variant thereof. In some embodiments, the system further comprises a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located 3' to the target deoxyribonucleic acid sequence. In some embodiments, the PAM sequence is located 5' to the target deoxyribonucleic acid sequence. In some embodiments, the PAM sequence includes any of SEQ ID NOS 46-66.

In some aspects, the disclosure provides a method of targeting an albumin gene, the method comprising introducing into a cell any of the systems described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to: a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 67-86. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the present disclosure provides a method of targeting a HAO1 gene or locus, the method comprising introducing into a cell any of the systems described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to: a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 611-633. In some embodiments, the guide ribonucleic acid sequence is configured to hybridize to: a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 615, 618, 620, 624 or 626. In some embodiments, the guide ribonucleic acid comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOS 645-684. In some embodiments, the guide ribonucleic acid comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOS 645-649, 652-656, 660-671, 674-675, or 681-684, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the targeting sequence of any of SEQ ID NOS 645-649, 652-656, 660-671, 674-675, or 681-684. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some embodiments, the present disclosure provides a method of disrupting a HAO-1 locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the HAO-1 locus, wherein the engineered guide RNA is configured to hybridize to or comprises: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO 611-626 or 627-633. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises a sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS 618, 620, 624, or 626, or a sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a targeting sequence of any of SEQ ID NOS 618, 620, 624, or 626. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 9 or table 12. In some embodiments, the cell is a mammalian cell. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the present disclosure provides a method of disrupting a TRAC locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRAC locus, wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NOs 139-158; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of SEQ ID NOs 119-138. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease includes any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises the fusion endonuclease having at least 55% identity to SEQ ID No. 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOS 121, 132, 136, 130, 134, 135, or 137 or a sequence that is at least 80% identical to a targeting sequence of any of SEQ ID NOS 121, 132, 136, 130, 134, 135, or 137. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7A. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some embodiments, the disclosure provides a method of disrupting a B2M locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (B) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the B2M locus, wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID nos. 185-210; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of SEQ ID NOs 159-184. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease includes any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOS 159, 165, 168, 174, or 184, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a targeting sequence of any of SEQ ID NOS 159, 165, 168, 174, or 184. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7B. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the disclosure provides a method of disrupting a TRBC1 locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC1 locus, wherein the engineered guide RNA is configured to hybridize to or comprises: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID nos. 252-292; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any one of SEQ ID NOs 211-251. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease includes any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA is a targeting sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOS: 211, 212, 215, 241, or 242 or comprising a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 211, 212, 215, 241, or 242. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7C. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the disclosure provides a method of disrupting a TRBC2 locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC2 locus, wherein the engineered guide RNA is configured to hybridize to or comprises: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NOs 338-382; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of SEQ ID NOs 293-337. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease is any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOS 296, 306, or 332 or comprises a targeting sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a targeting sequence of any of SEQ ID NOS 296, 306, or 332. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7C. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the present disclosure provides a method of disrupting an ANGPTL3 locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the ANGPTL3 locus, wherein the engineered guide RNA is configured to hybridize to or comprises: a targeting sequence having at least 80% identity to SEQ ID NOS 478-572; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any one of SEQ ID nos. 383-477. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease includes any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs 419, 425, 431, 439, 447, 453, 461, 467, 471, or 473, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7D. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the disclosure provides a method of disrupting a PCSK9 locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the PCSK9 locus, wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID No. 588-602; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of SEQ ID NOs 573-587. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease includes any of the fusion endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOs 574, 578, 581, or 585. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 7E. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some embodiments, the present disclosure provides a method of disrupting an albumin locus in a cell, the method comprising introducing into the cell: (a) Any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the albumin locus, wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOs 67-86 or 646-695; or wherein the engineered guide RNA comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the targeting sequence of any one of SEQ ID NOs 67-86 or 646-695. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the class 2, type II Cas endonuclease comprises any one of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease includes any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises any of the type II Cas endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID No. 10, or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotide of SEQ ID NO 722. In some embodiments, the engineered guide RNA comprises or is complementary to a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of SEQ ID NOs 67, 68, 70, 71, 72, 76, 79, 80, 647, 648, 649, 653, 654, 655, 656, 673, 680, 681 or 682. In some embodiments, the engineered guide RNAs comprise the nucleotide sequences of any one of the guide RNAs of table 6. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or the guide-polynucleotide or comprises contacting the cell with a Lipid Nanoparticle (LNP) comprising the RNP.

In some aspects, the present disclosure provides an endonuclease comprising an engineered amino acid sequence having at least 55% sequence identity to any one of SEQ ID NOS: 1-27, 108 or 109-110, or a variant thereof.

In some aspects, the present disclosure provides an engineered nuclease system comprising: endonucleases described herein; and an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the endonuclease is derived from an uncultured microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease,Cas14 endonuclease, cas12a endonuclease, cas12b endonuclease, cas12 c endonuclease, cas12d endonuclease, cas12e endonuclease, cas13a endonuclease, cas13b endonuclease, cas13c endonuclease, or Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease. In some embodiments, the system further comprises an MG ²⁺ Is a source of (a).

In some aspects, the present disclosure provides an engineered nuclease comprising: (a) A class II, type II Cas enzyme RuvC and HNH domain, or variants thereof, having at least 55% sequence identity to the RuvC and HNH domain of any one of SEQ ID NOs 1-27, 108 or 109-110; and (b) a class II, type II Cas enzyme PAM Interaction (PI) domain or variant thereof, the class II, type II Cas enzyme PI domain having at least 55% sequence identity to a PAM Interaction (PI) domain of any one of SEQ ID NOs 1-27, 108 or 109-110. In some embodiments, (a) and (b) do not naturally occur together. In some embodiments, the class II, type II Cas enzyme is derived from an uncultured microorganism. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease. In some embodiments, the engineered nuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs 1-27.

In some aspects, the present disclosure provides an engineered nuclease system comprising: an endonuclease according to any of the aspects or embodiments described herein; and an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of any one of SEQ ID NOS.28-32 or 33-44, or a variant thereof. In some embodiments, the system further comprises a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located 3' to the target deoxyribonucleic acid sequence. In some embodiments, the PAM sequence includes any of SEQ ID NOS 46-66.

In some embodiments, the present disclosure provides an engineered single molecule heterologous guide-polynucleotide that is compatible with a class II, class II enzyme according to any of the aspects or embodiments described herein, wherein the heterologous guide-polynucleotide comprises a chemical modification according to any of SEQ ID NOs 645-684.

In some aspects, the present disclosure provides a method of targeting an albumin gene, the method comprising introducing into a cell a system according to any one of the aspects or embodiments described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence comprising any one of SEQ ID NOs 67-86.

In some aspects, the present disclosure provides a method of targeting a HAO1 gene, the method comprising introducing into a cell a system according to any of the aspects or embodiments described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to any of SEQ ID NOs 611-633. In some embodiments, the guide ribonucleic acid sequence is configured to hybridize to any of SEQ ID NOs 615, 618, 620, 624, or 626. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOS: 645-684. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOS 645-649, 652-656, 660-671, 674-675, or 681-684.

In some aspects, the present disclosure provides cells comprising an endonuclease described herein. In some aspects, the present disclosure provides a cell comprising any of the nucleic acid molecules described herein. In some aspects, the present disclosure provides cells comprising the engineered nuclease systems described herein.

Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other different embodiments and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure/fig") ":

FIGS. 1A-1B depict the natural PAM specificity of the various effectors described herein. FIG. 1A shows a phylogenetic tree of the various effectors described herein. Fig. 1B is a PAM-specific table of natural RNA guided CRISPR-associated endonucleases.

Figure 2 illustrates the concept of domain exchange between RNA-guided CRISPR-associated nucleases.

Figures 3A and 3B depict an alignment of multiple sequences to guide the determination of the optimal breakpoint. Fig. 3A shows SaCas9 and SpCas9 aligned with several proteins described herein, and the terminal conserved residues (alanine residues) of these sequences were identified as the proposed C-terminal of the exchange segment. FIG. 3B depicts the C-terminal domain of the SaCas9 protein as the exchange span of the RuvC-III, WED, TOPO and CTD domains. PAM interactive domains consist of TOPO domains and CTD domains. The exchanged C-terminal domain does not contain active site residues (D10, E477 and H701 of RuvC domain and D556, D557 and N580 of NHN domain).

FIG. 4 depicts screening of chimeras by in vitro PAM enrichment assays when MG3-6 was recombined with various C-terminal domains from closely and remotely related nucleases. Sgrnas from the N-terminal parent domain are used for RNA-guided nuclease activity.

Fig. 5A-5B depict PAM sequences (fig. 5A) and Seq Logo depictions (fig. 5B) of PAM sequences of functional chimeras described herein. Given the breakpoint exchange of the predicted C-terminal domain of RuvC-III, WED, TOPO and CTD, the chimera is functional if recombined with a closely related nuclease. Even if the native protein is not functional in the same experiment, engineered chimeras tend to retain PAM specificity of the PAM interaction domain of the native protein.

FIG. 6 shows screening of chimeras by in vitro PAM enrichment assays in cases where the chimeras recombine MG3-6 with various c-terminal domains from closely and remotely related nucleases. Sgrnas from the C-terminal parent domain are used for RNA-guided nuclease activity. The numbers in brackets indicate sgRNA species. There was no rescue activity using sgrnas from the C-terminal parental domain.

FIG. 7 shows predicted structures of MG3-6 and MG 15-1. The WED and PI domains of MG3-6 were exchanged with the domains of the MG15-1 counterpart to generate chimera 1 (C1). Alternatively, the PI domain of MG3-6 is exchanged with the counterpart of MG15-1 to generate chimera 2 (C2).

Fig. 8A-8B depict PAM enrichment assay and sanger sequencing (Sanger sequencing) results in vitro for PAM specificity. C1: MG3-6+MG15-1 (WP) and C2: MG3-6+MG15-1 (P). Engineered chimeras tend to retain PAM specificity of the PAM interaction domain of the native protein. PAM enrichment assays were performed in triplicate. (FIG. 8A) shows an agarose gel description of an assay indicating that the sequence was cleaved in the presence of active enzyme, and (FIG. 8B) shows a Seqlogo description of the PAM sequence determined by the assay.

Figures 9A-9B depict the activity of the chimeras described herein in mammalian cells. mRNA encoding the chimera was co-transfected with 20 different sgRNAs (see, e.g., SEQ ID No: 67-86) into Hepa1-6 cells. Editing was assessed by sanger sequencing and CRISPR editing Inference (ICE). Fig. 9A shows the editing efficiency of the tested wizard. Two biological replicates are shown. FIG. 9B shows an insertion and deletion (indel) curve created by a representative wizard.

FIG. 10 depicts the results of guide screening in Hepa1-6 cells; the guide was delivered as mRNA and gRNA using lipofectamine Messenger Max.

FIG. 11A depicts structural parts of the MG3-6/3-4 guide. FIG. 11B depicts structural portions of the MG3-6 guide.

FIG. 12 depicts the activity of chemically modified MG3-6/3-4 guides in Hepa1-6 cells when using lipofectamine Messenger Max as mRNA and gRNA delivery.

FIG. 13 depicts the stability of the chemically modified MG3-6/3-4 guide at 37℃over 9 hours.

FIG. 14 depicts the stability of the chemically modified MG3-6/3-4 guide at 37℃over 21 hours.

FIGS. 15A-15B depict in vitro screening of V-A type chimeras. FIG. 15A depicts agarose gels of amplified cleavage products of each cleavage reaction. Positive enrichment was observed by the MG29-1+MG29-5 chimera, domain switching from the same family (numbers in brackets indicate sgRNA species). Fig. 15B depicts Seqlogo descriptions for PAM of parent enzyme and chimeras derived therefrom.

FIG. 16 depicts the results of gene editing of TRAC at the DNA level in HEK293T cells.

FIG. 17 depicts the results of gene editing of B2M at the DNA level in HEK293T cells.

FIG. 18 depicts the results of gene editing of TRAC at the DNA and phenotype level in T cells.

FIG. 19 depicts the results of gene editing of B2M at the DNA level in T cells.

FIG. 20 depicts gene editing results of TRBC1 and TRBC2 at the phenotypic level in T cells.

FIG. 21 depicts the results of gene editing of ANGPTL3 at the DNA level in Hep3B cells.

FIG. 22 depicts gene editing results of PCSK9 at the DNA level in Hep3B cells.

FIG. 23 depicts genome editing of MG3-6/3-4 at the HAO-1 locus in wild-type mice analyzed by next generation sequencing.

FIG. 24 depicts glycolate oxidase protein levels in the liver of mice treated with MG3-6/3-4mRNA and guide RNA targeting the HAO-1 gene.

FIG. 25 depicts genome editing at the HAO-1 locus in wild type mice treated with MG3-6/3-4mRNA and guide RNA 7 (G7) with 4 different chemical modifications that target HAO-1.

FIG. 26 depicts Western blot analysis of Glycolate Oxidase (GO) protein levels in the liver of 11 th day mice after treatment with LNP encapsulating MG3-6/3-4mRNA and sgRNA 7 (G7) with 4 different chemical modifications.

Brief description of the sequence Listing

The sequence listing filed herewith provides example polynucleotide and polypeptide sequences for use in methods, compositions and systems according to the present disclosure. The following is an example description of sequences therein.

MG3-6 chimera

SEQ ID NOS.1-27 show the full-length peptide sequences of MG3-6 chimeric nucleases.

SEQ ID NO. 108 shows the nucleotide sequence of an MG3-6/3-4 nuclease comprising a 5'UTR, NLS, CDS, NLS, 3' UTR and a polyA tail.

SEQ ID NOS.28-45 and 605-610 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 chimeric nucleases.

SEQ ID NOS.46-59 show the natural PAM specificity of the various effectors.

SEQ ID NOS.60-66 show the PAM specificity of the chimeric nucleases described herein.

SEQ ID NO. 603 shows the DNA coding sequence of MG 3-6/3-4.

SEQ ID NO. 604 shows the protein sequence of the MG3-6/3-4 cassette coding sequence.

MG29-1 chimera

SEQ ID NOS.109-110 show the full-length peptide sequences of MG29-1 chimeric nucleases.

SEQ ID NOS.111-113 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 chimeric nucleases.

SEQ ID NOS.114-116 show the natural PAM specificity of the various effectors.

SEQ ID NO 117 shows the PAM specificity of the chimeric nucleases described herein.

TRAC targeting

SEQ ID NOS.119-138 shows the nucleotide sequences of sgRNAs engineered to function with MG3-6/3-4 nucleases to target TRAC.

SEQ ID NOS 139-158 shows the DNA sequence of the TRAC target site.

B2M targeting

SEQ ID NOS.159-184 shows the nucleotide sequences engineered to function with MG3-6/3-4 nucleases in order to target the sgRNA of B2M.

The DNA sequence of the B2M target site is shown in SEQ ID NOS.185-210.

TRBC1 targeting

SEQ ID NOS.211-251 shows the nucleotide sequences of sgRNAs engineered to function with MG3-6/3-4 nucleases in order to target TRBC 1.

SEQ ID NOS.252-292 shows the DNA sequence of the TRBC1 target site.

TRBC2 targeting

SEQ ID NOS.293-337 shows the nucleotide sequences of sgRNAs engineered to function with MG3-6/3-4 nucleases in order to target TRBC 2.

SEQ ID NOS.338-382 shows the DNA sequence of the TRBC2 target site.

ANGPTL3 targeting

SEQ ID NOS: 383-477 shows the nucleotide sequence of an sgRNA engineered to function with MG3-6/3-4 nuclease in order to target ANGPTL 3.

SEQ ID NOS 478-572 shows the DNA sequence of the target site of ANGPTL 3.

PCSK9 targeting

SEQ ID NO 573-587 shows the nucleotide sequence of an sgRNA engineered to function with MG3-6/3-4 nuclease to target PCSK 9.

SEQ ID NO. 588-602 shows the DNA sequence of the PCSK9 target site.

Detailed Description

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Practice of some of the methods disclosed herein employs techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA unless otherwise indicated. See, e.g., sambrook and Green et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), 4 th edition (2012); cluster books "current guidelines for molecular biology experiments (Current Protocols in Molecular Biology) (edited by F.M. Ausubel et al); books "methods of enzymology (Methods In Enzymology) (Academic Press, inc.)," PCR 2: practical methods (PCR 2:A Practical Approach) (M.J.MacPherson, B.D.Hames and G.R.Taylor edition (1995)), harlow and Lane edition (1988) antibodies: laboratory manuals (Antibodies, A Laboratory Manual), animal cell culture: basic technology and specialty applications Manual (Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications), 6 th edition (R.I. Freshney edit (2010)), which is incorporated herein by reference in its entirety.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, where the term "include" or "having" or variations thereof is used in the detailed description or claims, such term is intended to be inclusive in a manner similar to the term "comprising".

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., limitations of the measurement system. For example, "about" may mean within one or more than one standard deviation in accordance with the practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

As used herein, "cell" generally refers to a biological cell. The cell may be the basic structure, function or biological unit of a living organism. The cells may be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaebacterial cells, cells of single cell eukaryotes, protozoa cells, cells from plants (e.g., from planted crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, tapioca, sugarcane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodium, goldfish algae, liverwort, moss cells), algae cells (e.g., botrytis cinerea (Botryococcus braunii), chlamydomonas reinharderia (Chlamydomonas reinhardtii), pseudomicroalga (Nannochloropsis gaditana), pyrenoidosa (Chlorella pyrenoidosa), c.agardh b. Horsetail algae (Sargassum c.agardh), etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit, spinosa, echinoderm, nematodes, etc.), cells from animals (e.g., fish, amphibians, reptiles, birds, rodents, animals (e.g., rats, mice, rats, etc.), non-human animals, rats, etc.). Sometimes, the cells are not derived from a natural organism (e.g., the cells may be synthetically manufactured, sometimes referred to as artificial cells).

As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may comprise ribonucleoside triphosphates, adenosine Triphosphate (ATP), uridine Triphosphate (UTP), cytosine Triphosphate (CTP), guanosine Triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may comprise, for example, [ αS ] dATP, 7-deaza-dGTP and 7-deaza-dATP, as well as nucleotide derivatives which confer nuclease resistance to the nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddntps) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to: ddATP, ddCTP, ddGTP, ddITP and ddTTP. The nucleotides may be unlabeled or detectably labeled, such as with a moiety comprising an optically detectable moiety (e.g., a fluorophore). The marks may also be made with quantum dots. The detectable label may comprise, for example, a radioisotope, a fluorescent label, a chemiluminescent label, a bioluminescent label, and an enzymatic label. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7' -dimethoxy-4 '5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N' -tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 4- (4 'dimethylaminophenylazo) benzoic acid (DABCYL), waterfall blue, oregon green, texas red, cyan, and 5- (2' -aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of the fluorescent-labeled nucleotide may include [ R6G ] dUTP, [ TAMRA ] dUTP, [ R110] dCTP, [ R6G ] dCTP, [ TAMRA ] dCTP, [ JOE ] ddATP, [ R6G ] ddATP, [ FAM ] ddCTP, [ R110] ddCTP, [ TAMRA ] ddGTP, [ ROX ] ddTTP, [ dR6G ] ddATP, [ dR110] ddCTP, [ dAMRA ] ddGTP and [ dROX ] ddTTP, which are available from platinum Alzheimer's company (Perkin Elmer, foster City, calif.); fluoLink deoxynucleotides, fluoLink Cy3-dCTP, fluoLink Cy5-dCTP, fluoroLink Fluor X-dCTP, fluoLink Cy3-dUTP and FluoLink Cy5-dUTP available from Amersham, arlington Heights, ill.) in Allington, ill; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2' -dATP, available from Boehringer Mannheim company (Boehringer Mannheim, indianapolis, ind.) of Indianapolis; and chromosome-labeled nucleotides available from Molecular Probes, eugenia, oreg, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, waterfall blue-7-UTP, waterfall blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, oreg green 488-5-dUTP, rhodamine green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, texas red-5-UTP, texas red-5-dUTP, and Texas red-12-dUTP. Nucleotides may also be labeled or tagged by chemical modification. The chemically modified mononucleotide may be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may comprise biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer generally to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof, in single-stranded, double-stranded or multi-stranded form. Polynucleotides may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any function. Polynucleotides may include one or more analogs (e.g., altered backbones, sugars, or nucleobases). Modification of the nucleotide structure, if present, may be imparted either before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholino, locked nucleic acids, glycerol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, cpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, plait-glycosides and Russian glycosides. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple loci (one locus), exons, introns, messenger RNAs (mRNA), transfer RNAs (tRNA), ribosomal RNAs (rRNA), short interfering RNAs (siRNA), short hairpin RNAs (shRNA), micrornas (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides comprising cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes and primers defined according to ligation assays. The nucleotide sequence may be interspersed with non-nucleotide components.

The term "transfection" or "transfected" generally refers to the introduction of a nucleic acid into a cell by a non-viral or viral-based method. The nucleic acid molecule may be a gene sequence encoding the whole protein or a functional part thereof. See, e.g., sambrook et al (1989), molecular cloning: laboratory Manual, 18.1-18.88.

The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to generally refer to a polymer of at least two amino acid residues joined by peptide bonds. This term does not denote a specific length of the polymer nor is it intended to suggest or distinguish whether the peptide was produced using recombinant techniques, chemical or enzymatic synthesis or naturally occurring. The term applies to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interspersed with non-amino acids. The term encompasses amino acid chains of any length, including full-length proteins as well as proteins with or without secondary or tertiary structures (e.g., domains). The term also encompasses amino acid polymers that have been modified; for example by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation, such as conjugation with a labeling component. As used herein, the terms "amino acids" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. The modified amino acids may comprise natural amino acids and unnatural amino acids that have been chemically modified to comprise groups or chemical moieties that do not naturally occur on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" encompasses D-amino acids and L-amino acids.

As used herein, "non-native" may generally refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein. Non-natural may refer to an affinity tag. Non-natural may refer to fusion. Non-naturally may refer to naturally occurring nucleic acid or polypeptide sequences that include mutations, insertions, or deletions. The non-native sequence may exhibit or encode an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that may also be exhibited by a nucleic acid or polypeptide sequence fused to the non-native sequence. The non-native nucleic acid or polypeptide sequence may be joined to a naturally occurring nucleic acid or polypeptide sequence (or variant thereof) by genetic engineering to produce a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.

As used herein, the term "promoter" generally refers to a regulatory DNA region that controls transcription or expression of a gene and may be located adjacent to or overlapping with a nucleotide or nucleotide region that initiates transcription of RNA. Promoters may contain specific DNA sequences that bind protein factors (commonly referred to as transcription factors) that promote binding of RNA polymerase to DNA, thereby resulting in transcription of the gene. A 'base promoter', also referred to as a 'core promoter', may generally refer to a promoter that contains all the essential elements that promote transcriptional expression of an operably linked polynucleotide. In some cases, the eukaryotic basal promoter comprises a TATA box or CAAT box.

As used herein, the term "expression" generally refers to the process of transcribing a nucleic acid sequence or polynucleotide (e.g., into mRNA or other RNA transcript) from a DNA template or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may comprise splicing of mRNA in eukaryotic cells.

As used herein, "operably linked," "operably linked," or grammatical equivalents thereof generally refers to the juxtaposition of genetic elements, such as promoters, enhancers, polyadenylation sequences, and the like, wherein the elements are in a relationship permitting them to operate in a desired manner. For example, a regulatory element, which may include a promoter or enhancer sequence, is operably linked to a coding region if the regulatory element helps to initiate transcription of the coding sequence. As long as this functional relationship is maintained, insertion residues will exist between the regulatory element and the coding region.

As used herein, "vector" generally refers to a macromolecule or association of macromolecules that includes or is associated with a polynucleotide and that can be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. Vectors typically include genetic elements, such as regulatory elements, operably linked to a gene to facilitate expression of the gene in a target.

As used herein, an "expression cassette" and a "nucleic acid cassette" are generally used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. In some cases, an expression cassette refers to a combination of a regulatory element and one or more genes that are operably linked for expression.

"functional fragment" of a DNA or protein sequence generally refers to a fragment that retains a biological activity (function or structure) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be its ability to affect expression in a manner attributed to the full length sequence.

As used herein, an "engineered" object generally indicates that the object has been modified by human intervention. According to a non-limiting example: nucleic acids may be modified by changing their sequence to a sequence that does not exist in nature; nucleic acids can be modified by ligating them to nucleic acids with which they are not associated in nature, such that the ligation product has a function that is not present in the original nucleic acid; the engineered nucleic acid can be synthesized in vitro using sequences that do not exist in nature; the protein may be modified by changing the amino acid sequence of the protein to a sequence that does not exist in nature; engineered proteins may acquire new functions or properties. An "engineering" system includes at least one engineering component.

As used herein, "synthetic" and "artificial" are used interchangeably to refer to a protein or domain thereof that has low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, the VPR and VP64 domains are synthetic transactivation domains.

As used herein, the term "tracrRNA" or "tracrRNA sequence" may generally refer to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% sequence identity or sequence similarity to a wild-type example tracrRNA sequence (e.g., tracrRNA or SEQ ID NO from streptococcus pyogenes, staphylococcus aureus (s. Aureus), etc.). tracrRNA may refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity or sequence similarity to a wild-type example tracrRNA sequence (e.g., tracrRNA from streptococcus pyogenes, staphylococcus aureus, etc.). tracrRNA may refer to a modified form of tracrRNA, which may include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations or chimeras. tracrRNA may refer to a nucleic acid that is at least about 60% identical in a stretch of at least 6 contiguous nucleotides to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from streptococcus pyogenes, staphylococcus aureus, etc.). For example, the tracrRNA sequence may be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild-type, exemplary tracrRNA sequence (e.g., tracrRNA from streptococcus pyogenes, staphylococcus aureus, etc.) over a stretch of at least 6 contiguous nucleotides. By identifying regions complementary to part of the repeat sequence in adjacent CRISPR arrays, a type II tracrRNA sequence can be predicted on genomic sequences.

As used herein, a "guide nucleic acid" may generally refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid may be RNA. The guide nucleic acid may be DNA. The guide nucleic acid may be programmed to site-specifically bind to the nucleic acid sequence. The nucleic acid or target nucleic acid to be targeted may comprise nucleotides. The guide nucleic acid may comprise nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of the double-stranded target polynucleotide that is complementary to and hybridizes to the guide nucleic acid may be referred to as the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and thus may not be complementary to the guide nucleic acid, may be referred to as the non-complementary strand. The guide nucleic acid may comprise a polynucleotide strand, and may be referred to as a "single guide nucleic acid". The guide nucleic acid may comprise two polynucleotide strands and may be referred to as a "bidirectional guide nucleic acid". The term "guide" may be included, if not otherwise stated, to refer to both single guide and double guide. The guide nucleic acid may include a segment that may be referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence. The nucleic acid targeting segment may comprise a sub-segment, which may be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment.

In the context of two or more nucleic acid or polypeptide sequences, the term "sequence identity" or "percent identity" generally refers to sequences that are identical or have the same specified percentage of amino acid residues or nucleotides when compared and aligned within a local or global comparison window to obtain maximum correspondence, e.g., in a pairwise alignment, or more (e.g., in a multiple sequence alignment), as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP adjusted using a parameter with a word length (W) of 3 and an expected value (E) of 10, a BLOSUM62 scoring matrix (gap penalty set to 11 present, extension 1) and using conditional composition scoring matrices for polypeptide sequences longer than 30 residues; BLASTs using parameters with word length (W) of 2, expected value (E) of 1000000, and PAM30 scoring matrix (for sequences less than 30 residues, gap penalty set to 9 to open the gap and 1 to extend the gap) (these are default parameters for BLASTs in BLAST suite available at https:// BLAST. CLUSTALW with parameters; smith-Waterman homology search algorithm with the following parameters: match 2, mismatch-1 and void-1; MUSCLE with default parameters; a MAFFT with the following parameters: the retree is 2 and the maximums is 1000; novafold with default parameters; HMMER hmmalign with default parameters.

As used herein, the term "ruvc_iii domain" generally refers to the third discontinuous segment of the RuvC endonuclease domain (RuvC nuclease domain comprises three discontinuous segments ruvc_ I, ruvC _ii and ruvc_iii). RuvC domains or segments thereof can generally be identified by alignment with recorded domain sequences, structural alignment with proteins with annotated domains, or by comparison with hidden markov models (Hidden Markov Model, HMM) constructed based on recorded domain sequences (e.g., pfam HMM PF18541 of ruvc_iii).

As used herein, the term "wedge" (WED) domain generally refers to a domain that interacts with predominantly the repetition of sgrnas and PAM duplex, such as found in Cas proteins, of anti-repeat duplex. WED domains can generally be identified by alignment with recorded domain sequences, structural alignment with proteins having annotated domains, or by comparison with Hidden Markov Models (HMMs) constructed based on recorded domain sequences.

As used herein, the term "PAM interaction domain" or "PI domain" generally refers to a domain that interacts with a protospacer proximity motif (PAM) outside of the seed sequence in the region targeted by the Cas protein. Examples of PAM interaction domains include, but are not limited to, topoisomerase homology (TOPO) domains and C-terminal domains (CTD) present in Cas proteins. PAM interaction domains or segments thereof can generally be identified by alignment with recorded domain sequences, structural alignment with proteins having annotated domains, or by comparison with Hidden Markov Models (HMMs) constructed based on recorded domain sequences.

As used herein, the term "REC domain" generally refers to a domain (e.g., present in a Cas protein) that includes at least one of two segments (REC 1 or REC 2) that are alpha helical domains that are considered to contact a guide RNA. REC domains or segments thereof can generally be identified by alignment with recorded domain sequences, structural alignment with proteins having annotated domains, or by comparison with a Hidden Markov Model (HMM) constructed based on recorded domain sequences (e.g., pfam PF19501 of domain REC 1).

As used herein, the term "BH domain" generally refers to a domain that is a bridged helix between NUC and REC leaves of a type II Cas enzyme (e.g., present in a Cas protein). BH domains or segments thereof can generally be identified by alignment with recorded domain sequences, structural alignment with proteins having annotated domains, or by comparison with Hidden Markov Models (HMMs) constructed based on recorded domain sequences (e.g., pfam PF16593 of domain BH).

As used herein, the term "HNH domain" generally refers to an endonuclease domain having characteristic histidine and asparagine residues. HNH domains can generally be identified by alignment with recorded domain sequences, structural alignment with proteins with annotated domains, or by comparison with Hidden Markov Models (HMMs) constructed based on recorded domain sequences (e.g., pfam HMM PF01844 of domain HNH).

The present disclosure includes variants of any of the enzymes described herein having one or more conservative amino acid substitutions. Such conservative substitutions may be made in the amino acid sequence of the polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions may be made by amino acid substitutions of similar hydrophobicity, polarity, and R chain length. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating mutated amino acid residues between the species (e.g., non-conservative residues that do not alter the essential function of the encoded protein). Such conservatively substituted variants can comprise variants that are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of the systems described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may encompass sequences with substitutions such that the activity of the critical active site residues of the endonuclease is not disrupted. In some embodiments, functional variants of any of the systems described herein lack substitution of at least one of the conserved residues or functional residues described herein. In some embodiments, functional variants of any of the systems described herein lack all of the conservative residues or functional residues described herein.

Conservative representations of providing functionally similar amino acids are available from various references (see, e.g., cright on, protein: structural and molecular Properties (Proteins: structures and Molecular Properties) (W H Frieman Press (W H Freeman & Co.); 2 nd edition (12 months 1993)). The following eight groups each contain amino acids that are conservatively substituted with each other:

a. alanine (a), glycine (G);

b. aspartic acid (D), glutamic acid (E);

c. asparagine (N), glutamine (Q);

d. arginine (R), lysine (K);

e. isoleucine (I), leucine (L), methionine (M), valine (V);

f. phenylalanine (F), tyrosine (Y), tryptophan (W);

g. serine (S), threonine (T); and

h. cysteine (C), methionine (M).

SUMMARY

The discovery of new Cas enzymes with unique functions and structures may provide the possibility to further disrupt deoxyribonucleic acid (DNA) editing techniques, thereby improving speed, specificity, function and ease of use. There are relatively few functionally characterized CRISPR/Cas enzymes in the literature relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microorganisms and the pure diversity of microbial species. This is in part because a large number of microbial species may not be readily cultivated under laboratory conditions. Metagenomic sequencing of natural environmental niches representing a large number of microbial species may provide the possibility of greatly increasing the number of new CRISPR/Cas systems recorded and accelerating the discovery of new oligonucleotide editing functions. A recent example of the success of this approach was demonstrated by the discovery of CasX/CasY CRISPR systems by metagenomic analysis of natural microbial communities in 2016.

The CRISPR/Cas system is an RNA-guided nuclease complex that has been described as acting as an adaptive immune system in microorganisms. In the natural environment of a CRISPR/Cas system, the CRISPR/Cas system appears in a CRISPR (clustered regularly interspaced short palindromic repeats) operon or locus, which typically comprises two parts: (i) An array of short repeated sequences (30-40 bp) separated by equally short spacer sequences encoding RNA-based targeting elements; and (ii) an ORF encoding a Cas encoding a nuclease polypeptide guided by an RNA-based targeting element and an accessory protein/enzyme. Efficient nuclease targeting of a particular target nucleic acid sequence typically requires both: (i) Complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a Protospacer Adjacent Motif (PAM) sequence within the defined vicinity of the target seed (PAM is typically a sequence that is not commonly represented within the host genome). CRISPR-Cas systems are generally classified into 2 categories, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity, depending on the exact function and organization of the system.

Class I CRISPR-Cas systems have large multi-subunit effector complexes and include I, III and type IV.

Type I CRISPR-Cas systems are considered to be of moderate complexity in terms of components. In a type I CRISPR-Cas system, an array of RNA targeting elements is transcribed into long precursor crrnas (pre-crrnas) that are processed at repeat elements to release short mature crrnas that direct nuclease complexes to nucleic acid targets when they are followed by a suitable short consensus sequence called a Protospacer Adjacent Motif (PAM). This treatment is performed by an endoribonuclease subunit (Cas 6) of a large endonuclease complex called cascade, which also includes the nuclease (Cas 3) protein component of the crRNA-guided nuclease complex. Cas I nucleases act primarily as DNA nucleases.

Type III CRISPR systems may be characterized by the presence of a central nuclease called Cas10 and a repeat-related mysterious protein (RAMP) comprising Csm or Cmr protein subunits. As in the type I system, mature crrnas are treated from pre-crrnas using Cas 6-like enzymes. Unlike type I and type II systems, type III systems appear to target and cleave DNA-RNA duplex (e.g., DNA strand that serves as a template for RNA polymerase).

Type IV CRISPR-Cas systems have an effector complex consisting of two genes of the RAMP proteins of the highly reduced large subunit nuclease (csf 1), cas5 (csf 3) and Cas7 (csf 2) groups and in some cases the genes of the predicted small subunits; such systems are typically found on endogenous plasmids.

Class II CRISPR-Cas systems typically have single polypeptide multi-domain nuclease effectors and include type II, type V and type VI.

Type II CRISPR-Cas systems are considered the simplest in terms of components. In a type II CRISPR-Cas system, the processing of a CRISPR array into a mature crRNA does not require the presence of a special endonuclease subunit, but rather requires a small trans-encoded crRNA (tracrRNA) region that is complementary to the array repeat sequence; the tracrRNA interacts with its corresponding effector nuclease (e.g., cas 9) and the repeat sequence to form a precursor dsRNA structure that is cleaved by endogenous rnase III, thereby generating a mature effector enzyme that loads both the tracrRNA and the crRNA. Cas II nucleases are noted as DNA nucleases. Type 2 effectors typically exhibit a structure consisting of RuvC-like endonuclease domains that employ an rnase H fold, wherein the fold of RuvC-like nuclease domains has an unrelated HNH nuclease domain inserted within. RuvC-like domains are responsible for cleavage of target (e.g., crRNA complementary) DNA strands, while HNH domains are responsible for cleavage of displaced DNA strands.

The V-type CRISPR-Cas system is characterized by a nuclease effector (e.g., cas 12) structure similar to that of a type II effector comprising RuvC-like domains. Similar to type II, most (but not all) V-type CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA; however, unlike type II systems, which require RNase III to cleave the pre-crRNA into multiple crRNAs, type V systems can use the effector nuclease itself to cleave the pre-crRNA. Like the type II CRISPR-Cas system, the type V CRISPR-Cas system is again noted as a DNA nuclease. Unlike the type II CRISPR-Cas system, some type V enzymes (e.g., cas12 a) appear to have strong single-stranded non-specific deoxyribonuclease activity activated by the first crRNA directed cleavage of a double-stranded target sequence.

Type VI CRIPSR-Cas system has RNA-guided RNA endonucleases. A single polypeptide effector of a type VI system (e.g., cas 13) includes two HEPN ribonuclease domains instead of a RuvC-like domain. Unlike type II and type V systems, type VI systems also do not appear to require tracrRNA to process pre-crRNA into crRNA in some embodiments. However, like the V-type system, some VI-type systems (e.g., C2) appear to have strong single-stranded non-specific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of the target RNA.

Because of the simpler architecture of class II CRISPR-Cas, it has been most widely used for engineering and development as a designer nuclease/genome editing application.

One of the early adaptations of such systems for in vitro use can be found in Jinek et al (Science) 2012, 8, 17, 337 (6096): 816-21, which is incorporated herein by reference in its entirety. Jink studies first describe a system that involves (i) recombinantly expressed, purified full-length Cas9 (e.g., class II, type II Cas enzymes) isolated from streptococcus pyogenes SF 370; (ii) Purified mature about 42nt crRNA with about 20nt 5 'sequence complementary to the target DNA sequence to be cleaved followed by a 3' tracr binding sequence (whole crRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence); (iii) In vitro transcription of purified tracrRNA from a synthetic DNA template carrying a T7 promoter sequence; and (iv) mg2+. Jinek later describes an improved engineering system in which the crRNA of (ii) is linked to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) capable of itself guiding Cas9 to a target.

Mali et al, (science.2013, month 2, 15; 339 (6121): 823-826), incorporated herein by reference in its entirety), later applied this system to mammalian cells by providing DNA vectors encoding the following: (i) An ORF encoding a codon optimized Cas9 (e.g., a class II, type II Cas enzyme) under a suitable mammalian promoter having a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., tkpa signal); and (ii) an ORF encoding an sgRNA (having a 5 'sequence starting with G followed by a 20nt complementary targeting nucleic acid sequence linked to a 3' tracr binding sequence, a linker and a tracrRNA sequence) under a suitable polymerase III promoter (e.g., U6 promoter).

Engineered nucleases

In some aspects, the disclosure relates to engineering of novel nucleic acid guided nucleases and systems. In some embodiments, the engineered nuclease is functional in a prokaryotic or eukaryotic cell for in vitro, in vivo, or ex vivo applications. In some embodiments, the present disclosure relates to engineering and optimization of systems, methods, and compositions for genome engineering involving sequence targeting, such as genome perturbation or gene editing, associated with nucleic acid-guided nuclease systems and components thereof.

In some aspects, the present disclosure provides engineered nucleases, which can comprise nucleic acid guided nucleases, chimeric nucleases, and nuclease fusions.

Chimeric or fusion engineered nucleases

A chimeric engineered nuclease as described herein can include one or more fragments or domains, and the fragments or domains can be orthologs of a nuclease, such as a nucleic acid-guided nuclease, a genus, species, or other phylogenetic group of organisms described herein. Fragments may be from nuclease orthologs of different species. The chimeric engineered nuclease may comprise fragments or domains from at least two different nucleases. The chimeric engineered nuclease may comprise fragments or domains of nucleases from at least two different species. The chimeric engineered nuclease may comprise fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleases or nucleases from different species. In some embodiments, a chimeric engineered nuclease comprises more than one fragment or domain from one nuclease, wherein the more than one fragment or domain is separate from a fragment or domain from a second nuclease. In some examples, the chimeric engineered nuclease comprises 2 fragments, each fragment from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 3 fragments, each fragment from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 4 fragments, each fragment from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 5 fragments, each fragment from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 3 fragments, wherein at least one fragment is from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 4 fragments, wherein at least one fragment is from a different protein or nuclease. In some examples, the chimeric engineered nuclease comprises 5 fragments, wherein at least one fragment is from a different protein or nuclease.

Ligation between fragments or domains from different nucleases or species may occur in multiple segments of unstructured regions. Unstructured regions may comprise regions exposed within the protein structure or regions that are not conserved within the various nuclease orthologs.

MG chimeric enzyme

CRISPR effectors described herein have native PAM specificity (see fig. 1). In one aspect, the present disclosure provides for the realization of novel PAM specificities through protein engineering. This novel PAM-specific realization can be achieved by RNA-guided domain exchange of CRISPR-associated nucleases (see fig. 2). Optimal breakpoints may exist during domain swapping and reassembly. The optimal breakpoint may be guided by the alignment of the multiple sequences described herein (see fig. 3).

In some aspects, the present disclosure provides a fusion endonuclease comprising: (a) An N-terminal sequence comprising a RuvC, REC, or HNH domain of a Cas endonuclease or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO 696; and (b) a C-terminal sequence comprising a WED, TOPO or CTD domain of a Cas endonuclease or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID nos. 697-721. In some embodiments, the fusion endonuclease comprises RuvC, REC, and HNH domains in (a). In some embodiments, the fusion endonuclease comprises RuvC and HNH domains in (a). In some embodiments, the fusion endonuclease comprises a WED, TOPO or CTD domain in (b). In some embodiments, the N-terminal sequence and the C-terminal sequence are not naturally present together in the same reading frame. In some embodiments, the N-terminal sequence and the C-terminal sequence are derived from different organisms. In some embodiments, the N-terminal sequence further comprises a RuvC-I, BH and RuvC-II domain. In some embodiments, the C-terminal sequence further comprises a PAM interaction domain. In some embodiments, the fused Cas endonuclease comprises a sequence that has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID nos. 1-27 or 108. In some embodiments, the fusion endonuclease is configured to bind to PAM (SEQ ID NO: 53) that is not nnRGGnT. In some embodiments, the fusion endonuclease is configured to bind to PAM comprising any of SEQ ID NOS: 46-52 or 54-66.

In some aspects, the disclosure provides an endonuclease comprising an engineered nucleic acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs 1-27, 108 or 109-110. In one aspect, the disclosure provides an endonuclease comprising an engineered nucleic acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs 8-12, 26-27 or 108. In one aspect, the present disclosure provides an engineered nuclease system comprising: endonucleases described herein; engineering of A guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and configured to bind to the endonuclease. In some embodiments, and the engineered guide ribonucleic acid sequence further comprises a tracr ribonucleic acid sequence. In some embodiments, the endonuclease is derived from an uncultured microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12 c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease. In some embodiments, the system further comprises Mg ²⁺ Is a source of (a).

In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) Any of the endonucleases described herein (e.g., fusion endonucleases) comprising (a) an N-terminal sequence comprising a RuvC, REC, or HNH domain of a Cas endonuclease or variant thereof having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:696, and (b) a C-terminal sequence comprising a RuvC, REC, or HNH domain of at least 99% sequence identity to any one of SEQ ID NOs: 697-721, the C-terminal sequence comprising at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 92%, to 92% A WED, TOPO or CTD domain of a Cas endonuclease or variant thereof that is 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity less; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising: a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence; wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence. In some embodiments, the endonuclease is derived from an uncultured microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12 c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease. In some embodiments, the system further comprises Mg ²⁺ Is a source of (a). In some embodiments, an endonuclease comprises a sequence that has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 8-12, 26-27, or 108. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 95%, at least 97%, at least with a non-degenerate nucleotide of any one of SEQ ID NOs 33, 34, 44, 45, 78, 84, or 87Sequences that are 98% or at least 99% sequence identity.

The systems of the present disclosure can be used in a variety of applications, such as nucleic acid editing (e.g., gene editing), binding to nucleic acid molecules (e.g., sequence-specific binding). Such systems can be used, for example, to address (e.g., remove or replace) genetic mutations that may cause disease in a subject, inactivate genes in order to determine their function in cells, as diagnostic tools for detecting pathogenic genetic elements (e.g., by cleaving retroviral RNAs or amplified DNA sequences encoding pathogenic mutations), as inactivating enzymes in combination with probes to target and detect specific nucleotide sequences (e.g., sequences encoding bacterial antibiotic resistance), inactivate viruses by targeting viral genomes or to fail to infect host cells, engineer organisms to produce valuable small molecules, macromolecules or secondary metabolites by adding genes or modifying metabolic pathways, create gene driven elements for evolutionarily selected as biosensors to detect foreign small molecules and nucleotide to cell interference.

Table A-selected sequences disclosed herein

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Examples

Example 1 plasmid

The chimeric sequences were codon optimized for E.coli (E.coli) expression via the Integrated DNA technologies company (Integrated DNA Technologies, IDT) website and synthesized and cloned into pET21 vectors at the Twist Bioscience company (Twist Bioscience), unless otherwise indicated. To construct pET21-MG3-6+MG15-1 (WP) and pET21-MG3-6+MG15-1 (P), the gene fragment was amplified from pMGX3-6 and pMGX15-1 using primer P441-P446. The resulting PCR product was purified by Zymo gel DNA recovery kit and assembled into pAL3 (digested by ClaI and XhoI) by NEBUilder HiFi DNA assembly. The DNA sequence of the cloned chimeric gene was confirmed by sanger sequencing service provided by the company elam biopharma.

EXAMPLE 2 bioinformatics analysis

Based on the presence of putative HNH and RuvC catalytic residues, CRISPR type II endonucleases used herein are predicted to have nuclease activity. In addition, structural predictions suggest that residues are involved in guiding, targeting, recognition and interaction with PAM. Based on the position of the important residues, predicted type II CRISPR endonuclease domains include three RuvC domains, HNH endonuclease domains, recognition domains, PAM interaction domains, and the like. For genomic sequences encoding full length type II endonucleases immediately adjacent to the CRISPR array, tracrRNA sequences engineered to be used as one-way guide RNAs by nucleases are predicted.

Multiple sequence alignments were performed on selected RNA-guided CRISPR type II endonuclease sequences using a built-in MUSCLE aligner on the Geneius Primer software (available at https:// www.geneious.com/prime) (see FIG. 3). Protein structures of MG3-6 and MG15-1 were predicted using DNASTAR NovaFold and shown by Protean 3D. Details of the chimeric compositions are shown in table 1. Protein variants that recognize non-canonical PAM are engineered by ligating domains from closely and remotely related type II CRISPR endonucleases under guidance through predicted structural model information and guide RNA optimization (see fig. 7).

TABLE 1 chimeric compositions

Example 3-in vitro PAM enrichment assay

The PAM sequence of the nuclease used herein was determined by expression in an escherichia coli lysate based expression system or in a recombinant in vitro translation (myTXTL, arbor biosciences (Arbor Biosciences) or PURExpress, new england biosciences (New England Biolabs)). The E.coli codon optimized protein sequence was transcribed and translated from the PCR fragment under the control of the T7 promoter. The mixture was diluted into a reaction buffer (10mM Tris pH 7.5, 100mM NaCl,10mM MgCl) with protein-specific sgRNA and PAM plasmid library (PAM library U67/U40) ₂ ) Is a kind of medium. The plasmid library contains spacer sequences that match those in a single guide, followed by 8N mixed bases, a subset of which is considered to have the correct PAM. After 1-3 hours, the reaction is stopped and the DNA is recovered by DNA cleaning kits such as Zymo DCC, AMPure XP beads, qiaquick, etc. Subjecting the DNA to a blunt end ligation reaction that adds an adapter sequence to the cleaved libraryThe plasmid remains intact while the circular plasmid remains intact. PCR was performed using primers specific for the library and adaptor sequences (LA 065 and LA 125) and resolved on the gel to identify active protein complexes (see fig. 4 and 6). The resulting PCR product was further amplified by PCR using high throughput sequencing primers (TrueSeq) and KAPA HiFi HotStart with cycle parameters 8. Samples subjected to NGS analysis were quantified by 4200tape station (agilent technologies (Agilent Technologies)) and pooled together. NGS library was purified by AMPure XP beads and quantified by KAPA library Quant kit (henna) kit using the AriaMx real-time PCR system (agilent technologies). Sequencing this library (a subset of the starting 8N library) revealed sequences containing the correct PAM (see fig. 5).

Example 4-unidirectional guide design for in vivo targeting

As used herein, unidirectional guide (sgRNA) structures include the following: 5'- -22nt protospacer-repeat-tracr- -3'. 20 one-way guides targeting mouse albumin intron 1 were designed using the Geneius Prime software (https:// www.geneious.com/Prime /). In some cases, the guide is chemically synthesized by the IDT and includes chemical modifications ("Alt-R" modifications) of the guide optimized by the IDT to improve the performance of the Cas9 guide.

Example 5 in vitro transcription of mRNA

The coding sequence (CDS) encoding the chimera (e.g., MG3-6+MG3-4 (SEQ ID NO: 10)) was codon optimized for mice and chemically synthesized at Twist bioscience. CDS was cloned into mRNA production vector pMG010. The architecture of pMG010 includes a series of elements: t7 promoter-5 'UTR-initiation codon-nuclear localization signal 1-CDS-nuclear localization signal 2-termination codon-3' UTR-107 nucleotide polyA tail (SEQ ID NO: 108). Plasmid pMG010 containing the MG3-6+MG3-4 CDS was purified from 200ml bacterial culture using the EndoFree plasmid kit (Qiagen). The vector was digested with SapI overnight to linearize the plasmid downstream of the polyA tail. Linearized vectors were purified using phenol/chloroform DNA extraction. In vitro transcription was performed using HiT 7T 7 RNA polymerase (new england biosystems) at 50 ℃ for 1 hour. The in vitro transcribed mRNA was treated with DNase at 37℃for 10 minutes and purified using MEGAclear transcription cleaning kit (ThermoFisher). mRNA was quantified by absorbance at 260nm and its size and purity were assessed by automatic electrophoresis (TapeStation, agilent) and demonstrated to have the expected size.

Example 6-Hepa1-6 cell transfection and Albumin targeting

300ng of mRNA and 350ng SEQ ID NO:67-86 of each one-way guide RNA (sgRNA) were co-transfected into Hepa1-6 cells as follows. The day before transfection, hepa1-6 cells were seeded into 24 wells at a density to achieve 70% confluence after 24 hours. The next day, 25 μl OptiMEM medium and 1.25 μ l Lipofectamine Messenger Max solution (Semerle Feisher) were mixed and vortexed for 5 seconds to make solution A. 300ng of MG3-6+MG3-4 chimeric mRNA and 350ng of one-way guide were mixed together with 25. Mu.l of OptiMEM in separate tubes to make solution B. Solutions a and B were mixed and incubated at room temperature for 10 minutes and then added directly to the Hepa1-6 cells. Media was aspirated two days after transfection and genomic DNA was purified according to instructions from Purelink genomic DNA mini-kit (sameifeishier inc) (see fig. 9). The results indicated that the best performing sgRNAs were those designated g87 (SEQ ID NO: 72) and g34 (SEQ ID NO: 70), with significant editing also occurring for the gRNAs g45 (SEQ ID NO: 67), g44 (SEQ ID NO: 71), g59 (SEQ ID NO: 76), g78 (SEQ ID NO: 68), g84 (SEQ ID NO: 79) and g33 (SEQ ID NO: 80).

EXAMPLE 7 Mulberry sequencing of genome editing samples

Primers flanking genomic regions targeted by single guide RNAs (e.g., albumin genes) were designed. PCR amplification using primers 57F (SEQ ID NO: 97) and 1072R (SEQ ID NO: 98) was performed using a Phusion fast high fidelity PCR master mix (Semer Fielder Co.) to yield 1016bp PCR products. The PCR products were purified and concentrated using a DNA cleaner and concentrator 5 (Zymo Research) and 100ng of the PCR products were subjected to Sanger sequencing (in the elen Biosciences) using 8pmol of separate sequencing primers (132F, 282F, 446R and 460F,SEQ ID NO:99-102). Mulberry sequencing results were analyzed using an algorithm called CRISPR edit inference (available at https:// gitsub. Com/synhego-open/ice) and data were plotted using GradPrsm (see FIG. 9B).

EXAMPLE 8 MG3-6/3-4 nuclease guide screening of mouse HAO-1 Gene Using mRNA transfection

Guide RNA targeting MG3-6/3-4 nuclease for exons 1 to 4 of the mouse HAO-1 gene (encoding glycolate oxidase) was identified on a computer by searching for the PAM sequence 3'NNAAA (A/T) N5'. A total of 23 guide chemistries with the least predicted off-target sites in the mouse genome were synthesized as one-way guide RNAs. 300ng mRNA and 120ng of one-way guide RNA were transfected into Hepa1-6 cells as follows. The day prior to transfection, hepa1-6 cells, which had been cultured in DMEM, 10% FBS, 1xNEAA medium for less than 10 days, were seeded into TC-treated 24 well plates without Pen/Strep. Cells were counted and a volume equivalent to 60,000 living cells was added to each well. Additional pre-equilibration medium was added to each well to bring the total volume to 500 μl. On the day of transfection, 25 μl of OptiMEM medium and 1.25ul Lipofectamine Messenger Max solution (Semerle Feisher) were mixed in the main mix, vortexed, and allowed to stand at room temperature for at least 5 minutes. In separate tubes, 300ng of MG 3-6-MG-3-4-encoded mRNA (SEQ ID NO: 108) and 120ng of sgRNA (scaffold sequence SEQ ID NO: 34) were mixed together with 25. Mu.L of OptiMEM medium and vortexed briefly. An appropriate volume of messenger max solution was added to each RNA solution, mixed by flicking the tube, and briefly spun at low speed. The complete editing reagent solution was incubated for 10 minutes at room temperature and then added directly to the Hepa1-6 cells. Two days after transfection, the medium was aspirated from each well of the Hepa1-6 cells and passed through a culture medium with MagMAX ^TM The genomic DNA was purified by automated magnetic bead purification from KingFisher Flex of the DNA multisample over 2.0 kit. The activities of the guides are summarized in tables 2 and 3, while the primers used are summarized in table 4.

Table 2: average Activity of MG3-6/3-4 guide at mouse HAO1 delivered by mRNA transfection

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Table 3: results of testing MG3-6/3-4 guide with a more relaxed PAM design at mouse HAO1 delivered by mRNA transfection

Table 4: primers designed for the mouse HAO1 gene were used for PCR and sanger sequencing at each of the first four exons.

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Example 9 guided chemical optimization of MG3-6/3-4 and MG3-6 II type nucleases

40 different chemically modified guides (named mAlb3634-34-0 to mAlb 3634-34-44) were designed and the activity of 39 of these guides was tested. Wizard, mH3634-34-32, failed rna synthesis and was therefore not tested. The guide spacer sequence chosen as a model for insertion of various chemical modifications was marb 3634-34 (targeting albumin intron 1) as it proved to be the most active guide in the guide screening of the mouse hepatocyte cell line Hepa1-6 cells (table 5 and fig. 10).

Table 5: activity of chemically modified wizards in Hepa1-6 cells

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The sgRNA of MG3-6/3-4 comprises a spacer at the 5' end followed by the CRISPR repeat and transactivation CRISPRRNA (tracr). The CRISPR repeat sequence and tracr are identical to the MG3-6 nuclease (FIGS. 11a, 11 b). CRISPR repeats and tracr form a structured RNA comprising 3 stem loops (fig. 11 a). The different regions of the stem-loop are modified by replacing the 2' hydroxyl group of ribose with a methyl group or by replacing the phosphodiester backbone with a Phosphorothioate (PS). In addition, the spacer at the 5' position of the guide is modified with 2' -O-methyl or a mixture of 2' -fluoro bases and PS bonds. Different combinations of chemical modifications were designed, called marb 3634-34-0 to marb 3634-34-44, and the sequences are shown in table 6.

39 single guide editing activities with identical base sequences but different chemical modifications were evaluated in Hepa1-6 cells by co-transfection of mRNA encoding MG3-6/3-4 and guide; the results are shown in table 6 and fig. 12.

Table 6: chemically modified MG3-6/3-4 guide sequences and Activity in Hepa1-6 cells when co-transfected with MG3-6/3-4mRNA

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A guide with the same base sequence and commercially available chemical modification called AltR1/AltR2 was used as a control. The spacer sequence in these guides targets the 22 nucleotide region in albumin intron 1 of the mouse genome. The guide mAlb3634-34-0 (without chemical modification) showed 72% activity relative to the AltR1/AltR2 guide. The wizard, marb 3634-34-1, showed 124% activity relative to the actr 1/actr 2 wizard, showing the importance of wizard stability for editing: the mAlb3634-34-1 is more stable than the mAlb3634-34-0 (FIGS. 13 and 14). Importantly, mAlb3634-34-17 retained 147% activity relative to AltR1/AltR 2. Incorporation of 2' -O-fluoro in the spacer greatly increased the stability of mAlb3634-34-35 and the guide retained 65% activity. mAlb3634-34-35 contains 2' -O-methyl and PS bonds in the loops of the three stem loops of the MG3-6/3-4 guide. Importantly, mAlb3634-34-42 retains 66% of its activity and this guide contains as much fluorine in the spacer as mAlb3634-34-17, but it also contains PS bonds in all the loops present in the gRNA. The activity of mAlb3634-34-27 was 67% and that of mAlb3634-34-29 was 114%. In the modification, these guides contained PS bonds in the loop of the first stem-loop and 2' -O-methyl in the first strand of the first stem-loop for mAlb3634-34-27 and mAlb3634-34-29, respectively. When these 2 modifications are combined (2' -O-methyl in the first strand of the first stem-loop and PS bond in the loop of the first stem-loop), the guide loses its activity (mAlb 3634-34-33, mAlb3634-34-36, mAlb 3634-34-38), showing the complexity of the gRNA/protein interaction and proving that the results of simple extrapolation are difficult to predict.

To test the stability of these chemically modified guides compared to the guide without chemical modification (natural RNA), stability assays were performed using crude cell extracts. The crude extract of mammalian cells is selected because it contains the nuclease mixture to which the guide RNA will be exposed when delivered to mammalian cells in vitro or in vivo. Hepa1-6 cells were collected by adding 3ml cold PBS to each 15cm dish and releasing the cells from the surface of the dish using a cell scraper. The cells were pelleted at 200g for 10 min and frozen at-80 ℃ for future use. For stability assays, cells were resuspended in 4 volumes of cold PBS (e.g., for 100mg pellet, cells were resuspended in 400ul of cold PBS). Triton X-100 was added to a concentration of 0.2% (v/v), the cells were vortexed for 10 seconds, placed on ice for 10 minutes, and vortexed again for 10 seconds. Triton X-100 is a mild nonionic detergent that disrupts cell membranes but does not inactivate or denature proteins at the concentrations used. A stable reaction was established on ice and included 20. Mu.l of crude cell extract, 2pmol each (1 ul in 2uM stock). Each wizard establishes six reactions, including: input, 0.5 hours, 1 hour, 4 hours, 9 hours, and in some cases 21 hours (time in hours refers to the length of time each sample was incubated). Samples were incubated at 37℃for 0.5 to 21 hours while the input control was placed on ice for 5 minutes. After each incubation period, the reaction was stopped by adding 300ul of a mixture of phenol and guanidine thiocyanate (Tri reagent, zymo research company), which immediately denatured all proteins and effectively inhibited ribonuclease and promoted subsequent recovery of RNA. After adding Tri reagent, the samples were vortexed for 15 seconds and stored at-20 ℃. RNA was extracted from the samples using the Direct-zol RNA miniprep kit (Zymo research company) and eluted in 100ul nuclease-free water. Detection of modified guides was performed using Taqman RT-qPCR using Taqman miRNA assay technology (Semer femto), and primers and probes were designed to specifically detect sequences in mAlb3634-34sgRNA, which was the same for all guides. The data are plotted as a function of the percentage of residual sgrnas associated with the input samples (tables 7 and 8; fig. 13 and 14).

Table 7: guide for chemical modification of MG3-6/3-4 stability at 37℃for 9 hours

Table 8: guide for chemical modification of MG3-6/3-4 stability at 37℃for 21 hours

Stability assays showed that the introduction of three 2' -O-methyl and three PS linkages at the 5' and 3' ends of the guide significantly improved stability (fig. 13 and 14). As in marb 3634-17 and marb 3634-42, the addition of additional 2' -fluoro to the 5' and 3' modifications did not show significant advantage at early time points (up to 9 hours), as shown in fig. 13, but a slight improvement in stability was apparent when the stability assay was run for 21 hours (fig. 14). The inclusion of 2-O-methyl and PS bonds (mAlb 3634-35) in all the loops of the stem loop provides a significant increase in stability compared to the guide with chemical modifications at the 5 'and 3' ends (mAlb 3634-1), as can be seen in FIG. 13. However, when these results are repeated and at a longer time point, this increase becomes less pronounced at an earlier time point and becomes pronounced at a longer time point of up to 21 hours, as can be seen in fig. 14. The inclusion of 2' -O-methyl in the first strand of the different stem loops provides stability advantages for up to 9 hours, as shown by comparing mAlb3634-0 with mAlb3634-29 and mAlb 3634-30. The combination of 2' -O-methyl in the first strand with all stem-loops and PS bond in the loops of all stem-loops, mAlb3634-36, showed significantly increased stability at 9 hours compared to the end-modified guide (mAlb 3634-0). However, this guide was inactive when tested by mRNA transfection in Hepa1-6 cells. In general, adding additional modifications (e.g., 2 '-O-methyl, 2' -O-fluoro, or PS linkages) to the end-modified guide does not provide a great advantage in terms of stability at an earlier time point (up to 9 hours) (fig. 13), but a small increase in stability at a longer time point is apparent (fig. 14). The large size (110 nt) and highly structured nature of such grnas may make them inherently more stable than shorter or less structured guide RNAs, limiting the stability benefits of chemical modification. The 5 'and 3' ends of the modification guide appear to provide a good level of protection against nucleases. However, adding additional modifications to the guide may provide further benefits in vivo, as these types of modifications may reduce immunogenicity.

Example 10 protein recombination of V-A type nuclease

To expand the ability to rapid PAM exchange beyond type II nucleases, three V-Sup>A nucleases were selected for protein recombination. Breakpoints are selected based on the predicted structural information (table 1). Similar to type II enzyme recombinants, type V chimeras show activity when proteins are recombined from closely related families. In vitro PAM enrichment and NGS analysis revealed consistent results: PAM of the chimera is inherited from the C-terminal parent. Potential structural breaks in protein recombination from distant related families can be avoided by using breakpoint optimization (fig. 15).

EXAMPLE 11 analysis of TRAC Gene editing results at the DNA level in HEK293T cells

MG3-6/4RNP (104 pmol protein/300 pmol guide) nuclei containing sgRNA described in Table 7A and SEQ ID NOS: 119-158 below were transfected into HEK293T cells (200,000) using a Lonza 4D electroporator. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing (fig. 16). The results indicate that sgrnas C1, F2 and B3 are most effective in inducing insertions and deletions, with significant editing also occurring for sgrnas D2, H2, A3 and C3.

Table 7A: gRNA and targeting sequences used in example 11

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EXAMPLE 12 analysis of the Gene editing results of B2M at the DNA level in HEK293T cells

MG3-6/4RNP (104 pmol protein/300 pmol guide) nuclei containing sgRNA described in Table 7B and SEQ ID NOS 159-210 below were transfected into HEK293T cells (200,000) using a Dragon 4D electroporators. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing (fig. 17). The results indicate that sgrnas a1, G1, B2, H2 and B4 are most effective in inducing editing, with significant editing also detected for sgrnas C1, D1, A2, H1, E2, F2, G2, A3, C3 and D3.

Table 7B: gRNA and targeting sequences used in example 12

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EXAMPLE 13 analysis of TRAC Gene editing results at the DNA and phenotype level in T cells

Primary T cells were purified from PMBC using a negative selection kit (meitian gentle (Miltenyi)) according to the manufacturer's recommendations. MG3-6/4RNP (104 pmol protein/120 pmol guide) nuclei containing sgRNA described in Table 7A and SEQ ID NOS: 119-158 were transfected into T cells (200,000) using a Dragon 4D electroporator. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing. For analysis by flow cytometry, 100,000T cells were stained with anti-CD 3 antibody at 4C for 30 min 3 days after nuclear transfection and analyzed on an Attune Nxt flow cytometer (fig. 18). The results show that sgrnas C1, D2, F2, H2, A3, B3, C3 and D3 show significant edits, with the largest edits made by sgrnas C1 and B3.

EXAMPLE 14 analysis of Gene editing results of B2M at the DNA level in T cells

Primary T cells were purified from PMBC using a negative selection kit (meitian gentle) according to the manufacturer's recommendations. MG3-6/4RNP (104 pmol protein/120 pmol guide) nuclei containing sgRNA described in Table 7B and SEQ ID NOS 159-210 were transfected into T cells (200,000) using a Dragon 4D electroporator. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing (fig. 19).

EXAMPLE 15 analysis of Gene editing results of TRBC1 and TRBC2 at the phenotypic level in T cells

Primary T cells were purified from PBMCs using a negative selection kit (meitian gentle) according to the manufacturer's recommendations. MG3-6/4RNP (104 pmol protein/120 pmol guide) nuclei containing sgRNA described in Table 7C and SEQ ID NOS 211-382 below were transfected into T cells (200,000) using a Lonza 4D electroporation apparatus. For analysis by flow cytometry, 100,000T cells were stained with anti-CD 3 antibody at 4C for 30 min 3 days after nuclear transfection and analyzed on an Attune Nxt flow cytometer (fig. 20). As can be seen from the results in fig. 20, for TRBC1, the sgrnas with the highest performance are A1, B1, E1, G4, H4, and B5. Similarly, for TRBC2, the highest performing sgrnas are D1, H1, and A5.

Table 7C: gRNA and targeting sequences used in example 15

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EXAMPLE 16 analysis of Gene editing results of ANGPTL3 at the DNA level in Hep3B cells

MG3-6/4RNP (104 pmol protein/120 pmol guide) nuclei containing sgRNA described in Table 7D and SEQ ID NOS: 383-572 below were transfected into Hep3B cells (100,000) using a Dragon 4D electroporator. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing (fig. 21). The results show that sgrnas E5, C6, A7, A8, A9, G10, E11, a12 and C12 are the highest performing sgrnas in this assay.

Table 7D: gRNA and targeting sequences used in example 16

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EXAMPLE 17 analysis of Gene editing results of PCSK9 at the DNA level in Hep3B cells

MG3-6/4RNP (104 pmol protein/120 pmol guide) nuclei containing sgRNA described in Table 7E and SEQ ID NOS 573-602 below were transfected into Hep3B cells (100,000) using a Dragon 4D electroporator. Cells were harvested and genomic DNA was prepared three days after transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify a single target sequence for each guide RNA. Amplicons were sequenced on a genecontaining MiSeq machine and analyzed with proprietary Python scripts to measure gene editing (fig. 22). The results show that sgrnas B1, F1, A2 and E2 achieved the highest editing performance, with significant editing also occurring for D2, C2, B2, H1 and F2.

Table 7E: gRNA and targeting sequences used in example 17

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EXAMPLE 18 in vivo Gene editing in mouse liver by systemic administration of lipid nanoparticle delivered chimeric nucleases MG3-6/3-4

To assess the ability of MG3-6/3-4 chimeric type II nucleases to edit the genome in vivo in living animals, lipid nanoparticles were used to deliver mRNA encoding MG3-6/3-4 nucleases (RNA version of SEQ ID NO: 603) and one-way guide RNA (sgRNA) targeting different portions of the coding sequence of the mouse HAO-1 gene (e.g., as described in the following tables). The HAO-1 gene encodes glycolate oxidase, an enzyme involved in glycolate metabolism, and is expressed mainly in hepatocytes in the liver. Screening of sgrnas targeting HAO-1 coding sequences was performed in the mouse hepatocyte line Hepa1-6 to identify active guides. Sgrnas mH364-7 and mH364-20, which exhibited 46% and 26% editing in Hepa1-6 cells when transfected with mRNA encoding MG3-6/3-4 nuclease, were selected for testing in mice. mH364-7 targets exon 2 and mH364-20 targets exon 4.

Various chemical modifications of the native RNA structure are incorporated into these sgrnas. When incubated in extracts from mammalian cells, the chemical modifications were selected based on their ability to improve the stability of sgrnas in vitro without negatively affecting editing activity. For initial testing in mice, sgrnas mH364-7 and mH364-20 incorporating chemistry 1 and chemistry 35 were selected for testing and designated mH364-7-1, mH364-20-1, mH364-7-35, mH364-20-35. The sequences of these guides containing chemical modifications are shown in table 9 below.

Table 9: sequence and chemical modification of guide RNAs tested in mice

m:2' -O methyl modified base,: phosphorothioate backbones

mRNA encoding MG3-6/3-4 nuclease was produced by in vitro transcription of linearized plasmid templates using T7 RNA polymerase, nucleotides and enzymes purchased from New England Biolabs or triple Biotechnology Co (Trilink Biotechnologies).

The DNA sequence transcribed into RNA (SEQ ID No: 603) comprises the following elements in the order 5 'to 3': t7 RNA polymerase promoter, 5' untranslated region (5 ' UTR), nuclear localization signal, short linker, coding sequence for MG3-6/3-4 nuclease, short linker, nuclear localization signal and 3' untranslated region, and about 100 nucleotides polyA tail (not included in SEQ ID No: 603).

The protein sequence encoded in the synthetic mRNA encoded in this MG3-6/3-4 cassette comprises the following 5 'to 3' elements: a nuclear localization signal from SV40, a five amino acid linker (GGGS), a protein coding sequence of MG3-6/3-4 nuclease from which the initiation methionine codon is removed, a 3 amino acid linker (SGG), and a nuclear localization signal from nucleoplasmin. The DNA sequence of the protein coding region of this cassette was modified using commercially available algorithms to reflect codon usage in humans. CleanCAP @ available from Triplex Biotechnology Co ^TM ) Reagents encoding approximately 100 nucleotide polyA tails in plasmids for in vitro transcription and co-transcriptionally end-cap mRNA. Uridine in mRNA was replaced with N1-methyl pseudouridine.

Lipid Nanoparticle (LNP) formulations for delivery of MG3-6/3-4mRNA and guide RNA are based on LNP formulations described in the literature, including Kauffman et al (Nano Lett) 2015,15,11,7300-7306

(https:// doi.org/10.1021/acs.nanolet.5b 024970). The four lipid components were dissolved in ethanol and mixed in the appropriate molar ratio to prepare a lipid working mixture. mRNA and guide RNA were mixed at a 1:1 mass ratio prior to formulation or formulated in LNP alone and then co-injected into mice at a 1:1 mass ratio of the two RNAs. In either case, the RNA was diluted in 100mM sodium acetate (pH 4.0) to prepare an RNA working stock solution. The lipid working stock and the RNA working stock were mixed in a microfluidic device (Ignite NanoAssembler, precision nanosystems company (Precision Nanosystems)) at a flow rate ratio of 1:3 and a flow rate of 12 ml/min, respectively. LNP was dialyzed against Phosphate Buffered Saline (PBS) for 2 to 16 hours and then concentrated using an Amicon spin concentrator (Millipore) until a reduced volume was reached. The concentration of RNA in LNP formulations was measured using Ribogreen reagent (Sesameisier). The diameter and Polydispersity (PDI) of the LNP were determined by dynamic light scattering. Representative LNPs range in diameter from 65nm to 120nm with PDI from 0.05 to 0.20.LNP was intravenously injected into 8 to 12 week old C57Bl6 wild-type mice (0.1 mL per mouse) via the tail vein at a total RNA dose of 1mg RNA per kg body weight. Eleven days after dosing, 3 out of each group of 5 mice were sacrificed and livers were collected and homogenized in digestion buffer provided in PureLink genomic DNA isolation kit (sameir femil) using a bead mill (ohni international (Omni International)). Genomic DNA was purified from the resulting homogenate using a PureLink genomic DNA isolation kit (Siemens technologies Co.) and quantified by measuring absorbance at 260 nm. Genomic DNA purified from buffer-only mice was used as a control. 28 days after dosing, the remaining 2 mice per group were sacrificed and livers were collected and homogenized using a bead mill (ohm corporation) in digestion buffer provided in PureLink genomic DNA isolation kit (zemoeimer). Genomic DNA was purified from the resulting homogenate using a PureLink genomic DNA isolation kit (Siemens technologies Co.) and quantified by measuring absorbance at 260 nm. Genomic DNA purified from buffer-only mice was used as a control.

The liver genomic DNA was then PCR amplified using a first set of primers flanking the two targeted regions of the guide. The PCR primers used are shown in table 10 below.

Table 10: sequences of PCR primers and next generation sequencing primers for analysis of genome editing in mice

The 5' end of these primers included a conserved region complementary to the PCR primer used in the second PCR, followed by 5N to provide sequence diversity and improve the quality of MiSeq sequencing, and ended with sequences complementary to the target region in the mouse genome. UsingA hot start high fidelity 2X master mix (New England Biolabs) PCR was performed on 100ng of genomic DNA and the annealing temperature was 60℃for a total of 30 cycles. Round 2 and 10 cycles of PCR were then performed using primers designed to add unique dual-cause nalmefene barcodes (IDTs) for next generation sequencing on the MiSeq instrument. Each sample was sequenced to a depth of greater than 10,000 reads using 150bp paired end reads. Reads were pooled to generate a single 250bp sequence, with the percent insertions and deletions and the insertion and deletion curves calculated using proprietary Python scripts.

For individual mice, NGS analysis results of insertions and deletions from mice on day 11 post-dosing are shown in table 11 and summarized in fig. 32.

Table 11: genome editing of MG3-6/3-4 at the HAO-1 locus in whole livers of wild-type mice analyzed by next generation sequencing on day 11 post LNP dosing.

Data for individual mice are shown. All mice receiving the guide RNA LNP also received LNP encapsulating MG3-6/3-4mRNA. The% of insertions and deletions of OOF is the percentage of all insertions and deletions of the sequence that results in the HAO1 coding sequence listing. The average total OOF% is the average percentage of all alleles of the HAO1 coding sequence box. The total number of NGS sequencing reads is given.

Group 2 mice received LNP encapsulated guide RNAmH 364-7-1. Group 3 mice received LNP encapsulated guide RNAmH 364-7-35. Group 4 mice received LNP encapsulating guide RNA mH 364-20-1. Group 5 mice received LNP encapsulated guide RNAmH 364-20-35. All mice in groups 2 to 5 also received LNP, which encapsulates MG3-6/3-4mRNA mixed with guide RNA containing 1:1RNA mass ratio prior to injection. No insertions and deletions were detected in the livers of PBS buffer injected mice (see table 11). Mice injected with LNP encapsulating guide 364mHA-G7-1 and MG3-6/3-4mRNA showed insertions and deletions at the target site in HAO-1 at an average frequency of 53.0%. Mice injected with LNP encapsulating guide 364mHA-G7-35 and MG3-6/3-4mRNA showed insertions and deletions at the target site in HAO-1 at an average frequency of 23.6%. Mice injected with LNP encapsulating guide 364mHA-G20-1 and MG3-6/3-4mRNA showed insertions and deletions at the target site in HAO-1 at an average frequency of 8.9%. Mice injected with LNP encapsulating guide 364mHA-G20-35 and MG3-6/3-4mRNA showed insertions and deletions at the target site in HAO-1 at an average frequency of 2.5%. These data demonstrate that guides with spacer 7 (364 mHA-G7-1 and 364 mHA-G7-35) are significantly more potent in vivo than guides with spacer 20 (364 mHA-G20-1 and 364 mHA-G20-35) when compared with guides with the same chemical modification. This is consistent with the higher editing level of these 2 guide sequences observed in the Hepa1-6 cells by mRNA-based transfection (mH 364-7 shows 46% insertions and deletions and mH364-20 shows 26% insertions and deletions in the Hepa1-6 cells). For both wizard 7 (for chemistry #1,2.2 times edit) and wizard 20 (for chemistry #1,3.5 times edit), wizard chemistry #1 resulted in a higher edit level than chemistry # 35. These data indicate that MG3-6/3-4 nuclease can be edited in vivo in mice at target sites specified by sgRNA. Furthermore, sgrnas with a set of chemical modifications designated chemical #1 were able to facilitate editing of 53% of genomic DNA in whole liver when delivered using LNP. The LNP used in these studies was absorbed by binding of apolipoprotein E (apoE) to LNP, a ligand that binds to low density lipoprotein receptor (see, e.g., yan et al, biochemical and biophysical research Comm (Biochem Biophys Res Commun) 2005 328 (1): 57-62.Doi:10.1016/j. Bbrc.2004.12.137, akinec et al, "molecular therapy (Mol Ther): 2010 (7): 1357-64, doi: 10.1038/mt.2010.85).

The liver is composed of many different cell types. In the liver of mice, hepatocytes account for approximately 52% of all cells (and 35% of hepatocytes contain two nuclei), with Coulomb cells (Kupffer cells) (18%), ito cells (8%) and endothelial cells (22%) constituting the remaining cells (histochemistry: cell biology (Histochem Cell Biol) & lt 131 & gt-726 https:// doi.org/10.1007/s 00418-009-0577-1). By extrapolation, without wishing to be bound by theory, it is predicted that about 60% [ ((52+ (0.35 x 52))/(48+ (0.35 x 52))) ] of total nuclei in mouse liver is derived from hepatocytes because LDL receptors are predominantly expressed on hepatocytes in the liver (see e.g. https:// www.proteinatlas.org/ENSG00000130164-LDLR/tissue/liver # imid_ 2815831), the LNP used in the mouse studies described herein is predicted to be predominantly absorbed by hepatocytes.

The HAO1 gene encodes the protein Glycolate Oxidase (GO), i.e., an intracellular enzyme involved in glycolate metabolism. To determine if gene editing observed in HAO1 gene resulted in reduced expression of GO protein in liver, total protein was extracted from individual leaves of mouse liver in the same study. The GO protein was detected using western blot assay using commercially available antibodies against mouse GO protein. Protein focal adhesion proteins were used as a loading control for western blots, as the predicted focal adhesion protein levels were not affected by gene editing of the HAO1 gene. As shown in fig. 24, the levels of GO protein in the liver of mice treated with LNP encapsulating MG3-6/3-4mRNA and the sho 1-targeted sgrnas were significantly reduced. Quantification of western blots using image analysis software, burle company ((Biorad)), and normalization of GO against focal adhesion protein levels showed an average 75%, 58%, 4% and 24% reduction in GO levels in mice treated with sgrnas mH364-7-1, mH364-7-35, mH364-20-1 and mH364-20-35, respectively. The extent of GO protein reduction correlated with the frequency of insertions and deletions in these groups of mice (see table 11). These data indicate that MG3-6/3-4 nucleases in combination with appropriately designed sgrnas can be used to create insertions and deletions in genes of interest in a living mammal and reduce (knock down) the production of proteins encoded by the genes. Reducing the expression of a particular gene may be therapeutically beneficial in a particular disease. In the case of HAO1 gene encoding GO protein, a decrease in GO protein levels in the liver is expected to be beneficial in patients with the primary hyperoxaluria type I of the genetic disease (Martin-Higueras, molecular therapy 24, 719-725). Thus, MG3-6/3-4 nuclease together with appropriate sgRNA containing appropriate chemical modifications targeting the HAO1 gene is a potential method of treating primary hyperoxaluria type I.

EXAMPLE 19 comparison of the efficiency of editing of the MG3-6/3-4 Gene in mice Using the same guide RNA sequence with four different chemical modifications

The effect of chemical modification on sgrnas on in vivo editing efficiency was further investigated by testing 4 different guide chemistries introduced into the same guide RNA sequence. Guide RNA7 targeting the mouse HAO1 gene was synthesized with chemical modifications #1, #35, #42, or # 45. The sequence of these wizards is shown in table 12 below.

Table 12: MG3-6/3-4sgRNA guide 7 sequence targeting mouse HAO1

mRNA encoding MG3-6/3-4 nuclease is produced by in vitro transcription of the linearized plasmid template using T7 RNA polymerase, nucleotides and enzymes purchased from New England Biolabs or triple Biotechnology. The DNA sequence transcribed into RNA comprises the following elements in 5 'to 3': t7 RNA polymerase promoter, 5' untranslated region (5 ' UTR), nuclear localization signal, short linker, coding sequence for MG3-6/3-4 nuclease, short linker, nuclear localization signal and 3' untranslated region (SEQ ID No: 603) and about 100 nucleotides polyA tail (not included in SEQ ID No: 603).

The protein sequence encoded in the synthetic mRNA encoded in this MG3-6/3-4 cassette comprises the following 5 'to 3' elements: a nuclear localization signal from SV40, a five amino acid linker (GGGS), a protein coding sequence of MG3-6/3-4 nuclease from which the initiation methionine codon is removed, a 3 amino acid linker (SGG), and a nuclear localization signal from nucleoplasmin. The DNA sequence of the protein coding region of this cassette was modified using commercially available algorithms to reflect codon usage in humans. CleanCAP @ available from Triplex Biotechnology Co ^TM ) Reagents encoding approximately 100 nucleotide polyA tails in plasmids for in vitro transcription and co-transcriptionally end-cap mRNA. Uridine in mRNA was replaced with N1-methyl pseudouridine. Lipid Nanoparticle (LNP) formulations for delivery of MG3-6/3-4mRNA and guide RNA are based on LNP formulations described in the literature, including Kauffman et al (nanometer flash, 2015,15,11,7300-7306, https:// doi. Org/10.1021/acs. Nanolet.5b024970). The four lipid components were dissolved in ethanol and mixed in the appropriate molar ratio to prepare a lipid working mixture. mRNA and guide RNA were mixed at a 1:1 mass ratio prior to formulation or formulated in LNP alone and then co-injected into mice at a 1:1 mass ratio of the two RNAs. In either case, the RNA was diluted in 100mM sodium acetate (pH 4.0) to prepare an RNA working stock solution. Lipid and RNA working stock solutions were prepared in a microfluidic device (Ignite NanoAssembler, precision nanosystems) at a flow rate ratio of 1:3 and 12 ml/min, respectivelyThe flow rates of the clocks are mixed. LNP was dialyzed against Phosphate Buffered Saline (PBS) for 2 to 16 hours and then concentrated using an Amicon spin concentrator (millbot corporation) until a reduced volume was reached. The concentration of RNA in LNP formulations was measured using Ribogreen reagent (Sesameisier). The diameter and Polydispersity (PDI) of the LNP were determined by dynamic light scattering. Representative LNPs range in diameter from 65nm to 120nm with PDI from 0.05 to 0.20.LNP was intravenously injected into 8 to 12 week old C57Bl6 wild-type mice (0.1 mL per mouse) via the tail vein at a total RNA dose of 1mg RNA per kg body weight. Ten days after dosing, 3 out of each group of 5 mice were sacrificed and livers were collected and homogenized using a bead mill (ohm corporation) in digestion buffer provided in PureLink genomic DNA isolation kit (sameir femoris). Genomic DNA was purified from the resulting homogenate using a PureLink genomic DNA isolation kit (Siemens technologies Co.) and quantified by measuring absorbance at 260 nm. Genomic DNA purified from buffer-only mice was used as a control. 28 days after dosing, the remaining 2 mice per group were sacrificed and livers were collected and homogenized using a bead mill (ohm corporation) in digestion buffer provided in PureLink genomic DNA isolation kit (zemoeimer). Genomic DNA was purified from the resulting homogenate using a PureLink genomic DNA isolation kit (Siemens technologies Co.) and quantified by measuring absorbance at 260 nm. Genomic DNA purified from buffer-only mice was used as a control.

The liver genomic DNA was then PCR amplified using a first set of primers flanking the two targeted regions of the guide. The PCR primers used are shown in table 10. The 5' end of these primers included a conserved region complementary to the PCR primer used in the second PCR, followed by 5N to provide sequence diversity and improve the quality of MiSeq sequencing, and ended with sequences complementary to the target region in the mouse genome. UsingA hot start high fidelity 2X master mix (New England Biolabs) was used to PCR 100ng of genomic DNA with an annealing temperature of 60℃and maintainedFor a total of 30 cycles. Round 2 and 10 cycles of PCR were then performed using primers designed to add unique dual-cause nalmefene barcodes (IDTs) for next generation sequencing on the MiSeq instrument. Each sample was sequenced to a depth of greater than 10,000 reads using 150bp paired end reads. Reads were pooled to generate a single 250bp sequence, with the percent insertions and deletions and the insertion and deletion curves calculated using proprietary Python scripts.

The compiled results are summarized in fig. 25 and listed in table 13.

Table 13: genome editing frequency in whole liver HAO1 Gene of Single mice treated with guide RNA7 encapsulating MG3-6/3-4mRNA and targeting HAO-1 Gene with chemical modifications 42 (mH 364-7-42), 45 (mH 364-7-45), 1 (mH 364-7-1) and 35 (mH 364-7-35)

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Control mice injected with PBS buffer contained no measurable insertions and deletions at the target site of guide 7. The mean insertion and deletion frequencies of LNP mice receiving guide mH364-7-1, mH364-7-35, mH364-7-42, and mH364-7-45 were 46.1%, 26.6%, 32.4%, and 32.1%, respectively, indicating that guide RNA chemistry #1 was the most potent, followed by #42 and #45, with chemistry #35 being the least potent. These data indicate that chemical modifications to the bases and backbones at the 5 'and 3' ends of the guide RNA provide the highest in vivo efficacy in the chemistry tested. Additional modification of internal bases did not improve in vivo efficacy. These findings are in contrast to published data for spCas9 sgRNA, where modifications of bases or backbones at both ends and at the internal sequence of the sgRNA are necessary for optimal in vivo editing (Yin et al, nature Biotechnology (Nature Biotechnology), doi: 10.1038/nbt.4005), and modifications of only the 5 'and 3' ends of the sgRNA use similar LNP delivery, achieving low levels of editing (20% insertions and deletions) in the liver.

Total RNA was purified from individual leaves of the same mice described in Table 13 and used to measure HAO-1mRNA levels by digital droplet PCR (dd-PCR). PBS-injected mice were used as controls and the levels of HAO-1mRNA in the liver of the edited mice were compared to these controls. dd-PCR assays are designed and optimized using standard techniques. ddPCR is a highly accurate method for determining the absolute copy number of a specific nucleic acid in a complex mixture (e.g., taylor et al, science report (Sci Rep), 7,2409 (2017). doi:10.1038/s41598-017-02217-x). Total liver RNA was first converted to cDNA by reverse transcription and then quantified in dd-PCR assay using GAPDH as an internal control to normalize between samples. As shown in Table 14, HAO1mRNA levels were reduced in individual mice treated with LNP encapsulating MG3-6/3-4mRNA and sgRNA targeting the mouse HAO1 gene, and the magnitude of the reduction correlated with the frequency of insertions and deletions.

Table 14: whole liver HAO1mRNA levels in individual mice treated with LNP encapsulating MG3-6/3-4mRNA and targeting guide RNA 7 with the chemically modified 42 (mH 364-7-42), 45 (mH 364-7-45), 1 (mH 364-7-1) and 35 (mH 364-7-35) genes.

The same mice in table 10 were analyzed.

A maximum reduction in HAO1mRNA was observed in the group of mice treated with sgRNA mH364-7-1, while a minimum reduction in HAO-1mRNA was observed in mice treated with sgRNA mH 364-7-35. When frameshift mutations are introduced into the coding sequence of the gene by a mechanism called nonsense-mediated decay, HAO1mRNA may be reduced (Brogna et al, nature Structure and molecular biology (Nat Struct Mol Biol), 16,107-113 (2009),doi:10.1038/ nsmb.1550). By MG3-6/34 observations of HAO-1mRNA reduction in mouse livers edited at HAO-1 gene are consistent with the presence of insertions and deletions that lead to high shift rates, as shown in table 15.

Table 15: analysis of the frequency of editing leading to frame shifts in the liver of mice treated with LNP encapsulating MG3-6/3-4mRNA and sgRNA No. 7 (G7) targeting the HAO-1 Gene

The percent of box (OOF%) is calculated by analyzing the NGS data using a custom algorithm.

In Table 15, the average frequency of insertions and deletions that result in the frameshift of the HAO1 coding sequence is determined from the NGS data. This analysis shows that most insertions and deletions result in frame shifts of all four tested sgrnas.

The HAO1 gene encodes the protein Glycolate Oxidase (GO), i.e., an intracellular enzyme involved in glycolate metabolism. To determine if gene editing observed in HAO1 gene resulted in reduced expression of GO protein in liver, total protein was extracted from individual leaves of mouse liver in the same study described in fig. 25 and tables 13 to 15. The GO protein was detected using western blot assay using commercially available antibodies against mouse GO protein. Equal amounts of protein were loaded onto western blots. As shown in FIG. 25, the levels of GO protein in the liver of mice treated with LNP encapsulating MG3-6/3-4mRNA and the sgRNA targeting HAO1 were reduced. Guides mH364-7-42 (mice 7, 8), mH364-7-45 (mice 12, 13) and mH364-7-1 (mice 17, 18) resulted in a significant reduction in GO protein. Among the 4 guides tested, guide mH364-7-35 (mice 22, 23) with the lowest level of insertions and deletions did not significantly reduce GO protein levels. These data indicate that MG3-6/3-4 nucleases in combination with appropriately designed sgrnas can be used to create insertions and deletions in genes of interest in a living mammal and reduce (knock down) the production of proteins encoded by the genes. Reducing the expression of a particular gene may be therapeutically beneficial in a particular disease. In the case of HAO1 gene encoding GO protein, a decrease in GO protein levels in the liver is expected to be beneficial in patients with the primary hyperoxaluria type I of the genetic disease (Martin-Higueras, molecular therapy 24, 719-725). Thus, MG3-6/3-4 nuclease together with appropriate sgRNA containing appropriate chemical modifications targeting the HAO1 gene is a potential method of treating primary hyperoxaluria type I.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited to the specific embodiments provided in the specification. While the invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be in a limiting sense. Numerous variations, changes, and substitutions will now be appreciated by those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention likewise encompasses any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the invention and their equivalents are therefore covered by this method and structure within the scope of these claims and their equivalents.

Sequence listing

<110> macrogenomics intellectual property technology Limited liability company (METAGENOMI IP TECHNOLOGIES, LLC)

<120> novel engineered and chimeric nucleases

<130> 55921-717.601

<140>

<141>

<150> 63/237,484

<151> 2021-08-26

<150> 63/140,620

<151> 2021-01-22

<160> 610

<170> patent In version 3.5

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Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

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Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

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Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

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Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

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Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

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Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

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Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

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Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

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Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

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Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

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Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

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Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

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Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

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Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

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Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

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Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

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Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

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Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

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Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

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Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

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Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

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Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

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Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

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Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

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Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

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Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

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Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

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Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

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Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

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Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

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Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

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Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

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Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

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Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

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Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

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Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

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Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

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His His Ala Val Asp Ala Ile Cys Ile Ser Phe Ser Arg Asp Phe Lys

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Tyr Asp Lys Glu Ile Lys Lys Asp Ile Ile Lys Gly Phe Asn Pro Glu

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Ile Val Lys Asn Ala Ile Asp Lys Ile Met Pro Tyr Pro Tyr Ala Asn

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Asp Lys Pro Phe Lys Gly Asn Thr Lys Pro Leu Glu Thr Ile Tyr Gly

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Leu Arg Thr Tyr Gly Asp Lys Ser Tyr Ile Thr Gln Arg Val Glu Leu

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Asn Ser Ile Asp Lys Lys Ala Thr Lys Ile Lys Ser Ile Ile Asp Glu

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Thr Ile Lys Asn Asp Leu Leu Asn Lys Leu Lys Glu Asn Pro Thr Glu

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Gln Glu Trp Lys Leu Met Leu Gln Asn Tyr Ile His Pro Lys Lys Gln

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Thr Lys Val Lys Lys Val Met Ile Ser Val Ser Glu Gly Glu Ile Thr

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Lys Asp Ser Asn Asn Arg Glu Arg Met Gly Glu Phe Val Asp Phe Gly

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Gln Ile Leu Tyr Phe Asn Glu Lys Gly Val Val Glu Val Met Pro Val

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Tyr Ser Asn Ile Lys Thr Thr Asp Val Lys Asp Lys Leu Gln Asn Met

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Gly Cys Lys Leu Tyr Asn Lys Gly Gln Met Phe Tyr Ser Gly Cys Leu

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Val Asp Ile Pro Lys Pro Phe Lys Ala Gly Ser Lys Glu Tyr Pro Ala

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Gly Arg Tyr Gln Ile Lys Thr Ile Arg Ser Asp Lys Val Ala Glu Leu

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Glu Asp Ala Cys Gly Asn Lys Ile Ser Thr Asn Val Lys Tyr Leu Val

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Pro Ala Glu Phe Lys Lys Val Glu Ser Lys

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Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

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Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

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Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

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Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

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Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

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Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

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Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

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Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

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Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

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Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

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Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

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Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

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Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

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Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

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Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

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Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

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Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

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Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

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Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

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Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

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Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

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Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

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Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

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Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

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Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

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Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

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Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

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Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

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Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

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Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

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Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Met Cys Ile Cys Phe Ala Pro Thr Ser Asn

740 745 750

Ala Lys Lys Ala Leu Ser Arg Lys Asn Ile Leu Pro Glu Glu Ile Ala

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Lys Asn Pro Glu Ser Asp Asp Ala Arg Asn Phe Phe Ala Lys Tyr Leu

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Ala Glu Val Val Pro Thr Lys Val Ala Ile Lys Lys Pro Glu Leu Glu

785 790 795 800

Gln Thr Ile Tyr Ser Lys Arg Val Ile Gly Gly Arg Gln Thr Ile Val

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Lys Lys Cys Asn Val Arg Asp Leu Ala Tyr Lys Gly Gln Asn Pro Lys

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Tyr Asp Phe Asp Thr Leu Thr Lys Arg Ile Lys Asp Ile Ile Asn Pro

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Val Ser Lys Arg Val Ile Glu Asp Phe Ala Lys Thr Glu Pro Thr Glu

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Ala Glu Trp Glu Asp Trp Cys Lys Tyr Glu Ala Ala Ile Pro Ser Lys

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Asn Gly Ser Pro Thr Arg Leu Leu Arg Val Leu Cys Lys Thr Lys Asp

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Asp Ala Glu Arg Phe Lys Asp Leu Ser Lys Asp Gly Cys Gly Ala Tyr

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Arg Lys Ser Lys Ser His Lys Gly Gln Phe Ile Trp Lys Asp Asn Lys

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Gly Asn Tyr Leu Val Ala Pro Val Tyr Ile Tyr Ser Ser Lys Gln Lys

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Val Tyr Ala Glu Leu Lys Asn Asn Pro Lys Cys Met Gly Ile Cys Asp

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Phe Phe Lys Thr Gly Cys Leu Val Lys Ile Ser Asn Glu Val Val Asp

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Glu Lys Lys Asn Arg Leu Trp Leu Lys Ala Gly Phe Tyr Asn Leu Asn

980 985 990

Ser Ile Ala Lys Glu Lys Arg Val Tyr Leu Thr Asp Val Asn Gly Gln

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Glu His Lys Lys Ile Pro Leu Gln His Leu Met Asn Ala Gly Met

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Lys Arg Val Glu Thr Asn Thr Ile

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Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

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Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

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Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

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Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

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Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

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Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

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Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

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Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

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Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

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Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

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Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

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Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

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Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Met Cys Leu Cys Phe Ala Pro Thr Gly Val

740 745 750

Asp Ser Arg Arg Ala Lys Leu Gly Glu Ile Leu Pro Glu Lys Leu Arg

755 760 765

Ser Glu Lys Ala Ala Arg Glu Phe Phe Lys Ser Tyr Leu Asp Lys Ile

770 775 780

Met Pro Val Asp Val Ala Pro Lys Lys Pro Arg Leu Glu Asp Gly Ile

785 790 795 800

Tyr Ser Lys Arg Ile Ile Gly Gly Lys Ala Cys Met Val Lys Arg Asn

805 810 815

Asn Leu Val Asp Leu Ala Tyr Lys Ser Gly Leu Lys Pro Val Phe Asp

820 825 830

Ile Pro Thr Leu Ile Lys Leu Val Asp Lys Lys Glu Lys Gly Ile Ile

835 840 845

Asn Pro Gln Ile Arg Lys Met Ile Gly Glu Phe Ala Ala Thr Asn Pro

850 855 860

Asp Glu Ser Ala Trp Arg Lys Trp Cys Glu Glu Val Arg Leu Pro Ser

865 870 875 880

Lys Ser Gly Leu Gly Ala Arg Val Leu Arg Val Leu Val Tyr Tyr Gly

885 890 895

Glu Ala Asp Glu Tyr Lys Asp Leu Ser Lys Asp Gly Cys Gly Ala Tyr

900 905 910

Arg Lys Gly Asp Gly His Lys Gly Gln Val Val Trp Glu Ser Val Asp

915 920 925

Gly Lys Tyr Tyr Val Glu Pro Val Tyr Val His Ala Ser Lys Ala Gly

930 935 940

Val Met Ala Ala Leu Asn Ala Asn Pro Lys Lys Lys Arg Ile Cys Gly

945 950 955 960

Met Phe Asn Ser His Cys Thr Val Asp Val Gly Asp Val Tyr Asn Asp

965 970 975

Arg Gly Asp Phe Ile Leu Pro Ala Gly Arg Tyr Met Val Asn Thr Ile

980 985 990

Leu Thr Thr Gly Arg Cys Val Leu Thr Asn Ala Asp Gly Glu Lys Arg

995 1000 1005

Asn Pro Ile Asn Ile Asn Tyr Leu Met Arg Ala Gly Met Arg Arg

1010 1015 1020

Val Glu Leu Ser Glu Leu

1025

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Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

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Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Met Cys Leu Cys Phe Ala Pro Thr Gly Val

740 745 750

Asn Ser Lys Arg Ala Arg Val Asp Met Leu Leu Pro Pro Lys Ile Arg

755 760 765

Ser Glu Lys Glu Ala Glu Leu Phe Phe Arg Lys Tyr Leu Asp Lys Leu

770 775 780

Ile Pro Val Asp Val Ala Pro Lys Lys Pro Lys Leu Glu Asp Gly Ile

785 790 795 800

Tyr Ser Met Arg Thr Val Gly Gly Lys Lys Ile Met Ala Arg Arg Val

805 810 815

Asn Leu Val Asp Leu Ala Tyr Lys Ser Gly Leu Lys Pro Val Tyr Asp

820 825 830

Val Ser Val Leu Ile Lys Leu Leu Asp Lys Lys Glu Arg Gly Ile Ile

835 840 845

Asn Pro Gln Ile Arg Lys Leu Val Ala Asp Phe Ala Arg Thr Asn Pro

850 855 860

Ser Glu Asp Glu Trp Lys Lys Trp Cys Gly Glu Cys Arg Leu Pro Ser

865 870 875 880

Lys Asn Gly Leu Gly Thr Arg Val Ile Arg Val Leu Leu Asn Tyr Gly

885 890 895

Glu Pro Ala Glu Tyr Lys Asp Leu Ser Lys Asp Gly Arg Gly Ala Phe

900 905 910

Arg Arg Gly Asp Gly His Lys Gly Gln Ile Val Trp Glu Ser Thr Asp

915 920 925

Gly Lys Tyr Cys Val Leu Pro Ile Tyr Val His Ala Ser Lys Ala Lys

930 935 940

Leu Leu Ala Glu Leu Cys Ala Asn Pro Lys Lys Lys Arg Ile Cys Gly

945 950 955 960

Ile Phe Thr Ser His Cys Met Val Lys Val Gly Asn Thr Tyr Asn Asn

965 970 975

Lys Gly Glu Leu Leu Leu Pro Glu Gly Val Tyr Met Leu Asn Thr Ile

980 985 990

Arg Thr Asp Gly Trp Ile Gln Leu Thr Ser Ala Asn Gly Asp Lys Ser

995 1000 1005

Lys Pro Ile Asn Ile Asn Tyr Leu Met Lys Ala Gly Met Lys Lys

1010 1015 1020

Val Pro Val Lys Asp Leu

1025

<210> 5

<211> 1068

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_2-4 chimera

<400> 5

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Leu Thr Leu Gly Leu Ala Thr Ala Leu Val

740 745 750

Pro Gly Ile Glu Arg Lys Glu Leu Arg Arg Ala Leu Ser Leu Arg Gln

755 760 765

Ala Lys Gly Asp Asp Ala Thr Leu Leu Arg Ser Asp Pro Lys Leu Gly

770 775 780

Glu Ala Leu Arg Trp Arg Thr Glu Asp Arg Phe Glu Ala Ala Pro Leu

785 790 795 800

Ser Gly Lys Leu Glu Ser Ala Val Arg Arg Ala Leu Ala Glu Gly Arg

805 810 815

Val Val Gln His Val Pro Ala Lys Arg Gln Gly Met Lys Val Asp Ser

820 825 830

Asn Phe Phe Gly Phe Val Glu Phe Asp Glu Thr Gly Arg Leu Arg Val

835 840 845

Arg Gln Lys Met Arg Ser Pro Thr Thr Arg Arg Arg Glu Ile Lys Thr

850 855 860

Thr Val Lys Asn Gly Lys Asn Leu His Thr Leu Ser His Leu Ser Leu

865 870 875 880

Asp Pro Lys Ser Trp Leu Gly Ala Pro Asp His Pro Leu Arg Arg Lys

885 890 895

Gln Leu Glu His Gly Leu Arg Thr Glu Asn Asp Leu Ala Asn Pro Lys

900 905 910

Leu Gly Asn Ile Arg Gly Met Leu Pro Ile Arg Glu Asn Trp Gly Ile

915 920 925

Ala Leu Ile Thr Lys Asp Gly Ser Pro Arg Leu Asp Val Ile Pro Tyr

930 935 940

Ile Asn Val His Gln Trp Leu Glu Val Leu Ala Leu Glu Asn Gly Gly

945 950 955 960

Gly Ser Pro Val Val Leu Arg Lys Gly His Leu Val Gly Phe Asp Ala

965 970 975

Glu Lys Cys Pro Glu Glu Tyr Cys Gly Ala Trp Met Leu Leu Gly Val

980 985 990

Lys Asp Gly Arg Ser Gly Thr Thr Leu Glu Leu Ile Arg Pro Trp Met

995 1000 1005

Val Ala Pro Arg Lys Gly Gly Thr Lys Glu Ser Ser Ala Lys Gln

1010 1015 1020

Ala Ile Lys Pro Ala Ser Gly Tyr Ser Glu Lys Glu Gly Lys Ala

1025 1030 1035

Ser Gly Val Phe Leu Gln Arg Ser Ala Asp Val Phe Leu Lys Leu

1040 1045 1050

Gly Leu Arg Pro Leu Asp His Asp Leu Thr Gly Ile Ala Ala Phe

1055 1060 1065

<210> 6

<211> 1006

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_2-7 chimera

<400> 6

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Val Thr Gln Gly Leu Ala Leu Leu Leu Phe

740 745 750

Ala Pro Glu Asp Trp Pro Leu Leu Val Lys Arg Asn Leu Pro Asp Ser

755 760 765

Glu Gln Arg His Leu Lys Ala Arg Tyr Pro Phe Leu Asp Phe Ser Ala

770 775 780

Asp Lys His Ile Ser Ile Gln Asp Leu Pro Glu Asp Thr Leu His Thr

785 790 795 800

Ile Ser Glu Arg Leu Ala Glu Cys Arg Val Val Arg His Ile Pro Ala

805 810 815

Lys Met His Gly Ile Ile Val Asp Gln Thr Thr Trp Gly Thr Val Ala

820 825 830

Ala Gly Ala Ile Thr Thr Leu Arg Gln Lys Thr Thr Glu Lys Asn Ala

835 840 845

Arg Cys Asp Glu Asn Gly Lys Arg Phe Ile Lys Thr Thr Glu Lys Lys

850 855 860

Arg Ser Leu Leu Leu Gly Gly Pro Asp Ala Pro Asp Gly Lys Leu Ala

865 870 875 880

Lys Ile Lys Gly Ala Ile Leu Val Thr Glu Asn Trp Gly Cys Ala Leu

885 890 895

Asp Pro Ser Pro Thr Val Ile Pro His Phe Lys Val Tyr Pro Gln Leu

900 905 910

Arg Ala Leu Arg Glu Lys Asn Gly Gly Arg Pro Ile Arg Ile Leu Arg

915 920 925

Lys Gly Ser Leu Ile Gln Val Lys Ala Gly Thr Tyr Gln Gly Ile Trp

930 935 940

Ser Val Ala Ser Ile Lys Asp Asn Ala Asp Gly Ile Cys Leu Asp Ile

945 950 955 960

Asn Ala Ala Asp Lys Val Lys Leu Glu Asn Arg Ser Asp Asp Ser Lys

965 970 975

Ile Asn Val Arg Leu Asp Ser Leu Arg Lys Ser Gly Leu Lys Ile Leu

980 985 990

Lys Pro Lys Leu Thr Gly Ala Cys Pro Thr Thr Ser Ser Pro

995 1000 1005

<210> 7

<211> 1109

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-1 chimera

<400> 7

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Leu Thr Leu Gln Ser Pro Ala Ile

740 745 750

Tyr Arg Val Leu Leu Thr Arg Val Asn Leu Lys His Glu His Glu Val

755 760 765

Thr Gly Glu Ala Pro Glu Trp Arg Asp Tyr Glu Gly Ala Asp Gln Ala

770 775 780

Glu Lys Val Leu Tyr Arg Arg Trp Gln Lys Asn Ile Ala Thr Leu Ala

785 790 795 800

Glu Leu Met Arg Gln Glu Ile Glu Asn Asn Arg Val Pro Val Thr Arg

805 810 815

Pro Ile Arg Leu Arg Lys Ser Arg Gly Ala Val His Asp Ala Thr Val

820 825 830

Met Lys Ala Leu Glu Arg Asp Leu Trp Gly Glu Trp Asp Ala Gln Ala

835 840 845

Ile Asp Arg Leu Val Asp Pro Glu Leu His Leu Ala Leu Arg Lys Leu

850 855 860

Phe Thr Ser Thr Lys Ser Lys Lys Ile Asp Val Asp Ala Thr Ser Gln

865 870 875 880

Gly Leu Pro Glu Arg Tyr Leu Ala Asn Gln Thr Val Gln Leu Phe Asp

885 890 895

Ala Asp Ala Pro Ser Val Met Ser Pro Arg Gly Ile Leu Arg Ile Gly

900 905 910

Ala Gly Thr His His Ala Arg Leu Leu Thr Trp Asp Asp Pro Lys Lys

915 920 925

Gly Pro Gln Leu Gly Ile Gln Arg Val Phe Ala Ala Glu Phe Gly Glu

930 935 940

Ile Leu Lys Asp Ala Ser Ser Asn Asp Leu Phe Glu Ala Pro Ile Pro

945 950 955 960

Phe His Thr Met Ser His Arg Asp Leu Gln Pro Lys Val Arg Ala Ala

965 970 975

Val Glu Gln Gly Leu Thr Arg Gln Ile Gly Trp Ile Thr Gln Gly Asp

980 985 990

Glu Leu Glu Ile Asp Pro Ala Asp Phe Val Gly Glu Ala Asn Ala Phe

995 1000 1005

Gly Asn Phe Leu Arg Glu Phe Pro Glu Arg Ser Trp Ser Ile Ala

1010 1015 1020

Gly Leu Lys Lys Ser Asn Thr Ile Val Ile Arg Pro Leu Leu Leu

1025 1030 1035

Ser Gln Glu Gly Val Thr Ala Ala Ile Ser Pro His Ala Ala Lys

1040 1045 1050

Ile Val Glu Asn Gly Ile Glu Leu Ser Asn Ser Thr Leu Phe Thr

1055 1060 1065

Ala Pro Gly Thr Gly Ile Ile Arg Arg Thr Gly Leu Gly Arg Pro

1070 1075 1080

Arg Trp Asp Ser Gly Pro Ala His Leu Pro Glu Ser Phe Asn Val

1085 1090 1095

His Ala Arg Met Thr Gln Gln Ser Ala Arg Asp

1100 1105

<210> 8

<211> 1122

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-2 chimera

<400> 8

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Leu Thr Leu Leu Asp Pro Ser Val

740 745 750

Ala Lys Thr Leu Ala Met Arg Leu Asp Leu Lys Arg Glu Gln Gln Asp

755 760 765

Ser Gly Arg Asp Thr Arg Trp Lys Glu Phe Lys Gly Leu Thr Pro Ala

770 775 780

Ser Gln Glu Arg Phe Ile Lys Trp Cys Gln Ala Ser Glu Cys Leu Ala

785 790 795 800

Asp Met Leu Arg Gln Gln Ile Glu Ala Asp Arg Val Pro Val Val Val

805 810 815

Pro Leu Arg Ile Ser Pro Ser Asn Gly Ala Val His Asp Asp Ser Val

820 825 830

Arg Pro Leu Thr Arg Gln Lys Ile Asp Ser Thr Trp Asp Arg Lys Ser

835 840 845

Ile Asn Arg Ile Val Asp Pro Glu Ile His Val Ala Met Arg Arg Leu

850 855 860

Leu Asn Asn Gly Thr Ser Leu Pro Glu Asp Lys Asn Arg Val Leu Asp

865 870 875 880

Leu Pro Asp Gly Asn Glu Leu Gly Pro His Asp Glu Val Glu Leu Phe

885 890 895

Ser Thr Ser Ala Ala Ser Ile Lys Leu Arg Arg Gly Gly Ser Ala Glu

900 905 910

Ile Gly Gly Ser Ile His His Ala Arg Val Tyr Ala Trp Met Gly Ala

915 920 925

Lys Gly Gln Leu Glu Tyr Gly Met Met Arg Val Phe Gly Ala Glu Phe

930 935 940

Pro Thr Leu Thr Lys Leu Ser Gly Ser Lys Asp Ile Leu Arg Met Pro

945 950 955 960

Ile His Ala Gly Ser Met Ser Tyr Arg Asp Met Gln Asp Arg Val Arg

965 970 975

Lys Pro Ile Glu Ser Asp Ile Ala Val Glu Leu Gly Trp Ile Thr Gln

980 985 990

Gly Asp Glu Leu Glu Ile Leu Pro Glu Ala His Leu Glu Thr Ala Gly

995 1000 1005

Gly Leu Gly Asp Phe Leu Lys Ser Phe Pro Glu Thr Gln Trp Thr

1010 1015 1020

Ile Asp Gly Phe Asn Asp Pro Ser Arg Leu Arg Val Arg Pro Arg

1025 1030 1035

Leu Met Ser Leu Glu Gly Arg Asp Thr Ile Asp Ala Met Gly His

1040 1045 1050

Leu Ser Asp Thr Glu Lys Leu Lys Ile Lys Gln Ala Leu Ser Lys

1055 1060 1065

Gly Leu Met Val Ser Ala Ser Glu Leu Leu Ser His Gly Ala Lys

1070 1075 1080

Ile Ile Arg Arg Asp His Leu Gly Arg Pro Arg Trp Arg Gly Asn

1085 1090 1095

Ala Arg Pro Val Ser Ile Glu Leu Glu Gln Val Ala Asn Gln Leu

1100 1105 1110

Val Asn His Arg Ser Val Asp Gly Gln

1115 1120

<210> 9

<211> 1125

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-3 chimeras

<400> 9

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Met Thr Leu Leu Asn Pro Ser Val

740 745 750

Ala Val Thr Leu Glu Gln Arg Arg Met Leu Lys Gln Glu Asn Asp Tyr

755 760 765

Ser Ser Pro Arg Gly Gln His Asp Asn Gly Trp Arg Asp Phe Ile Gly

770 775 780

Arg Gly Glu Ala Ser Gln Ser Lys Phe Leu His Trp Lys Lys Thr Ala

785 790 795 800

Val Val Leu Ala Asp Leu Ile Ser Glu Ala Ile Glu Gln Asp Thr Ile

805 810 815

Pro Val Val Asn Pro Leu Arg Leu Arg Pro Gln Asn Gly Ser Val His

820 825 830

Lys Asp Thr Val Glu Ala Val Leu Glu Arg Thr Val Gly Asp Ser Trp

835 840 845

Thr Asp Lys Gln Val Ser Arg Ile Val Asp Pro Asn Thr Tyr Ile Ala

850 855 860

Phe Leu Ser Leu Leu Gly Arg Lys Lys Glu Leu Asp Ala Asp His Gln

865 870 875 880

Arg Leu Val Ser Val Ser Ala Gly Val Lys Leu Leu Ala Asp Glu Arg

885 890 895

Val Gln Ile Phe Pro Glu Glu Ala Ala Ser Ile Leu Thr Pro Arg Gly

900 905 910

Val Val Lys Ile Gly Asp Ser Ile His His Ala Arg Leu Tyr Gly Trp

915 920 925

Lys Asn Gln Arg Gly Asp Ile Gln Val Gly Met Leu Arg Val Phe Gly

930 935 940

Ala Glu Phe Pro Trp Phe Met Arg Glu Ser Gly Val Lys Asp Ile Leu

945 950 955 960

Arg Val Pro Ile Pro Gln Gly Ser Gln Ser Tyr Arg Asp Leu Ala Ala

965 970 975

Thr Thr Arg Lys Phe Ile Glu Asn Gly Gln Ala Thr Glu Phe Gly Trp

980 985 990

Ile Thr Gln Asn Asp Glu Ile Glu Ile Ser Ala Glu Glu Tyr Leu Ala

995 1000 1005

Thr Asp Lys Gly Asp Ile Leu Ser Asp Phe Leu Gly Ile Leu Pro

1010 1015 1020

Glu Ile Arg Trp Lys Val Thr Gly Ile Glu Asp Asn Arg Arg Ile

1025 1030 1035

Arg Leu Arg Pro Leu Leu Leu Ser Ser Glu Ala Ile Pro Asn Met

1040 1045 1050

Leu Asn Gly Arg Leu Leu Thr Gln Glu Glu His Asp Leu Ile Ala

1055 1060 1065

Leu Val Ile Asn Lys Gly Val Arg Val Val Val Ser Thr Phe Leu

1070 1075 1080

Ala Leu Pro Ser Thr Lys Ile Ile Arg Arg Asn Asn Leu Gly Ile

1085 1090 1095

Pro Arg Trp Arg Gly Asn Gly His Leu Pro Thr Ser Leu Asp Ile

1100 1105 1110

Gln Arg Ala Ala Thr Gln Ala Leu Glu Gly Arg Asp

1115 1120 1125

<210> 10

<211> 1134

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-4 chimera

<400> 10

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Met Thr Leu Leu Asn Arg Ser Val

740 745 750

Ala Leu Thr Leu Glu Gln Arg Ser Gln Leu Arg Arg Ala Phe Tyr Glu

755 760 765

Leu Glu Leu Asp Lys Leu Asp Arg Asp Gln Leu Lys Pro Gly Glu Asp

770 775 780

Trp Arg Asn Phe Thr Gly Leu Tyr Glu Ala Ser Gln Asn Lys Phe Ser

785 790 795 800

Glu Trp Lys Lys Ala Ala Thr Val Leu Gly Asp Leu Leu Ala Glu Ala

805 810 815

Ile Glu Asp Asp Ala Ile Ala Val Val Ser Pro Leu Arg Leu Arg Pro

820 825 830

Gln Asn Gly Ser Val His Asp Asp Thr Ile Asn Ala Val Lys Lys Leu

835 840 845

Thr Leu Gly Ser Ala Trp Pro Ala Asp Ala Val Lys Arg Ile Val Asp

850 855 860

Pro Glu Ile Tyr Leu Ala Met Lys Asp Val Leu Gly Lys Leu Lys Glu

865 870 875 880

Leu Pro Glu Asp Ser Ala Arg Ser Leu Glu Leu Ser Asp Gly Arg Tyr

885 890 895

Ile Glu Ala Asp Asp Glu Val Leu Phe Phe Pro Lys Lys Ala Ala Ser

900 905 910

Ile Leu Thr Pro Arg Gly Ala Ala Glu Ile Gly Asn Ser Ile His His

915 920 925

Ala Arg Leu Tyr Ser Trp Leu Thr Lys Lys Gly Glu Leu Lys Phe Gly

930 935 940

Met Leu Arg Val Tyr Gly Ala Glu Phe Pro Trp Leu Met Arg Glu Ser

945 950 955 960

Gly Ser Arg Asp Val Leu His Met Pro Ile His Pro Gly Ser Gln Ser

965 970 975

Phe Arg Gly Met Gln Asp Gly Val Arg Lys Ala Val Glu Ser Gly Glu

980 985 990

Ala Val Glu Phe Gly Trp Ile Thr Gln Asp Asp Glu Leu Glu Phe Asp

995 1000 1005

Pro Glu Asp Tyr Ile Ala His Gly Gly Asp Asp Glu Leu Asn Arg

1010 1015 1020

Leu Leu Arg Val Met Pro Glu Arg Arg Trp Arg Val Asp Gly Phe

1025 1030 1035

Tyr Asn Ala Gly Thr Leu Arg Ile Arg Pro Ala Leu Leu Ser Ala

1040 1045 1050

Glu Gln Leu Pro Ser Glu Leu Gln Lys Lys Val Ala Asp Lys Thr

1055 1060 1065

Leu Ser Asp Val Glu Leu Ile Leu Leu Arg Ala Val Gln Arg Gly

1070 1075 1080

Leu Phe Val Ala Ile Ser Ser Phe Leu Pro Leu Glu Ser Leu Lys

1085 1090 1095

Val Ile Arg Arg Asn Asn Leu Gly Phe Pro Arg Trp Arg Gly Asn

1100 1105 1110

Gly Asn Leu Pro Thr Ser Phe Glu Val Arg Ser Ser Ala Leu Arg

1115 1120 1125

Ala Leu Gly Val Glu Gly

1130

<210> 11

<211> 1123

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-7 chimera

<400> 11

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Leu Thr Leu Leu Asn Arg Ser Val

740 745 750

Ala Val Thr Leu Glu Gln Arg Arg Leu Ile Lys Gln Gln Arg Glu Tyr

755 760 765

Ser Leu Glu Lys Ser Arg Arg Glu Arg Asp Asn Val Trp Arg Asp Phe

770 775 780

Met Gly Leu Gly Pro Ala Ala Gln Glu Lys Phe Ala Lys Trp Lys Lys

785 790 795 800

Thr Ala Tyr Val Leu Ala Asp Ile Ile Lys Glu Ala Ile Ser Asn Asp

805 810 815

Ala Ile Pro Val Val Ser Pro Leu Arg Leu Arg Pro Gln Asn Gly Ser

820 825 830

Val His Leu Asp Thr Val Asp Ala Val Leu Glu Arg Thr Ile Gly Asp

835 840 845

Ala Trp Thr Val Asp Gln Val His Arg Ile Val Asn Pro Gln Ile Tyr

850 855 860

Leu Ala Phe Ala Gly Tyr Leu Gly Asn Gln Lys Ala Leu Asp Pro Asp

865 870 875 880

Ser Ser Arg Val Leu Ala Leu Asn Asp Gly Arg Lys Leu Thr Ala Glu

885 890 895

Asp Val Ile Tyr Val Phe Pro Glu Lys Ala Ala Ser Ile Leu Thr Pro

900 905 910

Arg Gly Val Val Lys Ile Gly Glu Ser Val His His Val Arg Leu Tyr

915 920 925

Ala Trp Lys Asn Arg Lys Gly Lys Ala Glu Val Gly Met Leu Arg Val

930 935 940

Phe Gly Ala Glu Phe Pro Trp Leu Met Arg Glu Ser Gly Val Lys Asp

945 950 955 960

Val Leu Arg Val Pro Ile His Thr Gly Ser Gln Ser Tyr Arg Asp Leu

965 970 975

Ser Phe Thr Val Arg Lys Asn Ile Glu Lys Gly Glu Ala Ala Glu Ile

980 985 990

Gly Trp Leu Thr Gln Asn Glu Glu Leu Glu Phe Asn Pro Glu Ser Tyr

995 1000 1005

Leu Gln Glu Gly Gly Lys Asp Lys Leu Ala Lys Phe Leu Ala Phe

1010 1015 1020

Leu Pro Glu Thr Arg Trp Arg Val Asp Gly Phe Pro Met Pro Asp

1025 1030 1035

Lys Leu Arg Ile Arg Pro Ala Leu Leu Ser Arg Glu Glu Ile Pro

1040 1045 1050

Glu Gly Val Phe Arg Thr Glu Glu Gln Ser Leu Leu Glu Glu Ala

1055 1060 1065

Leu Thr Lys Gly Leu Ile Ile Ala Thr Lys Gly Leu Leu Ser Leu

1070 1075 1080

Pro Asp Val Lys Val Leu Arg Arg Asn Asn Leu Gly Ile Pro Arg

1085 1090 1095

Trp Arg Gly Gly Ser Tyr Arg Pro Val Ser Leu Asp Ile Gln Arg

1100 1105 1110

Ala Ala Leu Ala Ala Leu Asp Glu Gln Glu

1115 1120

<210> 12

<211> 1134

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_3-8 chimera

<400> 12

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Met Thr Leu Leu Asn Arg Ser Val

740 745 750

Ala Leu Thr Leu Glu Gln Arg Ser Gln Leu Arg Arg Ala Phe Tyr Glu

755 760 765

Gln Gly Leu Asp Lys Leu Asp Arg Asp Gln Leu Lys Pro Glu Glu Asp

770 775 780

Trp Arg Asn Phe Ile Gly Leu Ser Leu Ala Ser Gln Glu Lys Phe Leu

785 790 795 800

Glu Trp Lys Lys Val Thr Thr Val Leu Gly Asp Leu Leu Ala Glu Ala

805 810 815

Ile Glu Asp Asp Ser Ile Ala Val Val Ser Pro Leu Arg Leu Arg Pro

820 825 830

Gln Asn Gly Arg Val His Lys Asp Thr Ile Ala Ala Val Lys Lys Gln

835 840 845

Thr Leu Gly Ser Ala Trp Ser Ala Asp Ala Val Lys Arg Ile Val Asp

850 855 860

Pro Glu Ile Tyr Leu Ala Met Lys Asp Ala Leu Gly Lys Ser Lys Val

865 870 875 880

Leu Pro Glu Asp Ser Ala Arg Thr Leu Glu Leu Ser Asp Gly Arg Tyr

885 890 895

Leu Glu Ala Asp Asp Glu Val Leu Phe Phe Pro Lys Asn Ala Ala Ser

900 905 910

Ile Leu Thr Pro Arg Gly Val Ala Glu Ile Gly Gly Ser Ile His His

915 920 925

Ala Arg Leu Tyr Ser Trp Leu Thr Lys Lys Gly Glu Leu Lys Ile Gly

930 935 940

Met Leu Arg Val Tyr Gly Ala Glu Phe Pro Trp Leu Met Arg Glu Ser

945 950 955 960

Gly Ser His Asp Val Leu Arg Met Pro Ile His Pro Gly Ser Gln Ser

965 970 975

Phe Arg Asp Met Gln Asp Thr Thr Arg Lys Ala Val Glu Ser Ser Glu

980 985 990

Ala Val Glu Phe Ala Trp Ile Thr Gln Asn Asp Glu Leu Glu Phe Glu

995 1000 1005

Pro Glu Asp Tyr Ile Ala His Gly Gly Lys Asp Glu Leu Arg Gln

1010 1015 1020

Phe Leu Glu Phe Met Pro Glu Cys Arg Trp Arg Val Asp Gly Phe

1025 1030 1035

Lys Lys Asn Tyr Gln Ile Arg Ile Arg Pro Ala Met Leu Ser Arg

1040 1045 1050

Glu Gln Leu Pro Ser Asp Ile Gln Arg Arg Leu Glu Ser Lys Thr

1055 1060 1065

Leu Thr Glu Asn Glu Ser Leu Leu Leu Lys Ala Leu Asp Thr Gly

1070 1075 1080

Leu Val Val Ala Ile Gly Gly Leu Leu Pro Leu Gly Thr Leu Lys

1085 1090 1095

Val Ile Arg Arg Asn Asn Leu Gly Phe Pro Arg Trp Arg Gly Asn

1100 1105 1110

Gly Asn Leu Pro Thr Ser Phe Glu Val Arg Ser Ser Ala Leu Arg

1115 1120 1125

Ala Leu Gly Val Glu Gly

1130

<210> 13

<211> 1039

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_4-2 chimera

<400> 13

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Val Ala Ile Ala Leu Thr Asp Pro Ala Ala

740 745 750

Leu Lys Ser Ile Ser Gln Ala Ala Ser Asp Glu Arg Arg Gly Gly Arg

755 760 765

Val Ser Phe Gly Ala Val Ala Leu Pro Trp Val Asp Phe Ile Gly Asp

770 775 780

Val Gln Ala Ala Ile Glu Ala Ile Asn Val Ser His Arg Pro Ser Arg

785 790 795 800

Lys Val Asn Gly Ala Leu His Glu Glu Thr Phe Tyr Gly Pro Arg Gly

805 810 815

Met Asp Gly Asp Gly Arg Pro Thr Gly Tyr Val Gln Arg Lys Pro Val

820 825 830

Glu Arg Leu Ser Ala Lys Glu Ile Pro Asn Ile Pro Asp Pro Ala Val

835 840 845

Arg Glu Ala Val Gln Ala Lys Leu Asp Glu Val Gly Gly Thr Pro Ala

850 855 860

Gln Ala Phe Lys Asp Pro Ala Asn His Pro Val Arg Lys Arg Gly Ile

865 870 875 880

Pro Val His Lys Val Arg Leu Arg Leu Asn Ile Asn Pro Val Gln Val

885 890 895

Gly Ser Gly Ala Thr Glu Arg His Val Leu Thr Gly Ser Asn His His

900 905 910

Met Glu Ile Ile Glu Val Arg Asp Ala Lys Gly Gly Lys Lys Trp Thr

915 920 925

Gly Arg Leu Val His Arg Leu Glu Ala Lys Arg Arg Ala Leu Gly Arg

930 935 940

Glu Thr Ile Val Asp Arg Ala Val Gln Ala Gly Arg Gln Phe Gln Phe

945 950 955 960

Ser Leu Ser Pro Gly Asp Met Ile Glu Leu Thr Gly Glu Asp Gly Glu

965 970 975

Arg Lys Leu His Val Val Arg Ser Ile Ser Glu Gly Arg Ile Glu Tyr

980 985 990

Val Asp Ala Arg Asp Ala Arg Lys Lys Ala Asp Ile Arg Ala Ser Gly

995 1000 1005

Asp Trp Arg Lys Pro Ala Val Gly Ser Leu Leu Arg Leu His Cys

1010 1015 1020

Arg Lys Val Val Val Thr Pro Phe Gly Glu Ile Arg Tyr Ala Asn

1025 1030 1035

Asp

<210> 14

<211> 1051

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_4-5 chimera

<400> 14

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Val Val Ile Ala Leu Thr Gly Pro Gly Thr

740 745 750

Val Gln Ala Leu Thr Arg Ala Ala Leu Arg Ala Lys Glu Leu Gly Arg

755 760 765

Arg Leu Phe Val Pro Leu Asp Pro Pro Trp Ala Asp Arg Asp Ser Phe

770 775 780

Leu Arg Asp Val Arg Ala Ser Val Glu Ala Ile Thr Val Ser Tyr Arg

785 790 795 800

Val Asp Arg Lys Val Ser Gly Gln Leu His Glu Glu Ser Asn Tyr Ser

805 810 815

Lys Pro His Met Thr Val Asp Asn Lys Gly Asn Leu Val Glu His Arg

820 825 830

His Ile Arg Lys Pro Leu Lys Asp Met Ser Val Glu Glu Val Glu Ala

835 840 845

Ile Val Asp Asp Arg Val Arg Lys Leu Val Gln Glu Lys Leu Arg Gln

850 855 860

Leu Gly Gln Glu Pro Lys Lys Ala Phe Ala Asp Glu Ala Asn His Pro

865 870 875 880

Tyr Phe Thr Thr Ala Asp Gly Arg Leu Val Pro Ile His Lys Ala Arg

885 890 895

Ile Arg Lys Thr Val Ala Thr Ile Thr Val Gly Pro Pro Gln Cys Pro

900 905 910

Arg His Val Ala Pro Gly Leu Asn His His Ile Glu Ile Leu Ala Val

915 920 925

Arg Asp Pro Ala Gly Ala Val Thr His Trp Glu Gly Glu Leu Val Ser

930 935 940

Leu Phe Glu Ala Ala Arg Arg Val Lys Ala Gly Glu Pro Val Val Arg

945 950 955 960

Arg Asn His Gly Pro Asn Lys Asp Phe Leu Phe Ser Leu Ala Lys Gly

965 970 975

Glu Tyr Val Glu Met Glu Leu Gln Pro Gly Lys Arg Gln Leu Phe Arg

980 985 990

Val Thr Val Ile Ser Ala Lys Gln Ile Glu Phe Arg Leu His His Asp

995 1000 1005

Ala Arg Pro Thr Met Leu Leu Arg Lys Thr Pro Gly Ala Arg Val

1010 1015 1020

Ile Arg Ser Pro Gly Ser Leu Phe Lys Ala Lys Ala Arg Lys Val

1025 1030 1035

Ala Val Asp Pro Leu Gly Asn Val Phe Pro Ala Asn Asp

1040 1045 1050

<210> 15

<211> 1061

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_6-3 chimeras

<400> 15

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ile Val Val Ala Phe Thr Asn Arg Ser Thr

740 745 750

Leu Lys Arg Leu Ser Asp Glu Asn Lys Arg Ile Gly Thr Ala Glu Trp

755 760 765

Met Asp Ala Asp Glu Ser Gly Arg Ala Thr Asn Asp Glu Ile Lys Arg

770 775 780

Arg Leu Gly Gly Arg Ile Asp Leu Ser Glu Pro Trp Pro Thr Phe Arg

785 790 795 800

Asn Asp Val Glu Val Ser Ile Asn Asn Ile Thr Val Ser His Arg Val

805 810 815

Asn Arg Lys Val Ser Gly Ala Leu His Glu Glu Thr Tyr Tyr Gly Pro

820 825 830

Thr Asp Glu Pro Ala Pro Lys Asn Lys Glu Met Met Val Leu Arg Lys

835 840 845

Ser Val His Gln Leu Ser Lys Lys Asp Leu Gly Leu Ile Arg Asp Glu

850 855 860

Thr Ile Arg Gln Ile Val Asn Asp Glu Val Gln Lys Arg Met Asp Asn

865 870 875 880

Gly Glu Ser Gln Ala Asn Ala Ile Ala Ser Leu Glu Ala Asp Pro Pro

885 890 895

Phe Ile Ile Ser Pro Lys Ala Lys Val Pro Ile Arg Lys Val Arg Leu

900 905 910

Leu Met Lys Lys Asp Pro Gln Ile Met His Tyr Phe Glu Asn Lys Asn

915 920 925

Gly Glu Glu Asp Arg Ala Ala Leu Tyr Gly Asn Asn His His Ile Ala

930 935 940

Ile Tyr Glu Thr Ser Asp Lys Asn Gly Val Lys Lys Gln Ile Gly Ile

945 950 955 960

Val Ile Pro Met Met Glu Ala Ala Arg Arg Val Lys Asp Gly Asp Pro

965 970 975

Ile Val Met Lys Asp Tyr Arg Pro Asp His Thr Phe Leu Tyr Ser Leu

980 985 990

Ala Lys Asn Asp Met Ile Phe Asn His Glu Asp Glu Gln Ile Tyr Arg

995 1000 1005

Val Gln Lys Ile Asn Ser Asp Gly Thr Ile Met Phe Arg Gln Asn

1010 1015 1020

Asn Val Ala Met Lys Gly Gln Ser Asp Pro Gly Val Tyr Phe Lys

1025 1030 1035

Ser Gly Ser Arg Leu Gly Ala Ser Lys Ile Lys Ile Ser Pro Ile

1040 1045 1050

Gly Glu Ile Phe Pro Ala Asn Asp

1055 1060

<210> 16

<211> 990

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_14-1 chimera

<400> 16

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Cys Val Ile Ala Ala Cys Ser Pro Ser Leu

740 745 750

Val Ile Lys Thr Ala Arg Ile Asn Gln Glu Thr His Trp Ser Ile Thr

755 760 765

Arg Gly Met Asn Glu Thr Gln Arg Arg Asp Ala Ile Met Lys Ala Leu

770 775 780

Glu Ser Val Met Pro Trp Glu Thr Phe Ala Asn Glu Val Arg Ala Ala

785 790 795 800

His Asp Phe Val Val Pro Thr Arg Phe Val Pro Arg Lys Gly Lys Gly

805 810 815

Glu Leu Phe Glu Gln Thr Val Tyr Arg Tyr Ala Gly Val Asn Ala Gln

820 825 830

Gly Lys Asp Ile Ala Arg Lys Ala Ser Ser Asp Lys Asp Ile Val Met

835 840 845

Gly Asn Ala Val Val Ser Ala Asp Glu Lys Ser Val Ile Lys Val Ser

850 855 860

Glu Met Leu Cys Leu Arg Leu Trp His Asp Pro Glu Ala Lys Lys Gly

865 870 875 880

Gln Gly Ala Trp Tyr Ala Asp Pro Val Tyr Lys Ala Asp Ile Pro Ala

885 890 895

Leu Lys Asp Gly Thr Tyr Val Pro Arg Ile Ala Lys Ala His Thr Gly

900 905 910

Arg Lys Ala Trp Lys Pro Val Pro Glu Ser Ala Met Ala Lys Pro Pro

915 920 925

Leu Glu Ile Tyr Phe Gly Asp Leu Val Gln Ile Gly Asp Phe Ile Gly

930 935 940

Arg Phe Ser Gly Tyr Asn Ile Asn Asn Ala Asn Trp Ser Phe Thr Asp

945 950 955 960

Arg Leu Thr Arg Leu Asn Leu Ser Cys Pro Thr Val Gly Gln Leu Asn

965 970 975

Asn Asp Leu Ser Pro Val Val Ile Arg Glu Ser Pro Ile Lys

980 985 990

<210> 17

<211> 1096

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_15-1 chimera

<400> 17

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Val Ile Ile Ala Cys Ala Thr Gln Gly Ile

740 745 750

Val Asn Lys Val Ser Arg Tyr Ser Lys Ser Arg Glu Leu Trp Asp Tyr

755 760 765

Glu Val Asp Met Glu Thr Gly Glu Val Leu Gln Lys Lys Asn Lys Asn

770 775 780

Thr Lys Asp Val Phe Pro Glu Pro Trp Leu Asn Phe Arg Tyr Glu Leu

785 790 795 800

Glu Gln Lys Val Arg Val Arg Pro Leu Asp Ile Pro Glu Thr Ala Asp

805 810 815

Ile Thr Glu Met Glu Glu Pro Phe Val Ser His Met Pro Asn Arg Lys

820 825 830

Ile His Gly Pro Ala His Lys Glu Thr Ile Arg Ser Gly Arg Leu Lys

835 840 845

Glu Glu Gly Tyr Thr Ile Ser Lys Thr Ala Leu Ile Asp Leu Lys Leu

850 855 860

Thr Glu Asp Lys Glu Glu Ile Lys Gly Tyr Tyr Asn Lys Glu Ser Asp

865 870 875 880

Arg Leu Leu Tyr Glu Ala Leu Lys Lys Gln Leu Gln Arg Tyr Gly Gly

885 890 895

Lys Ala Lys Glu Ala Phe Lys Glu Pro Phe His Lys Pro Lys Ala Asp

900 905 910

Gly Thr Pro Gly Pro Ile Val Asn Lys Val Lys Ile Met Glu Lys Ser

915 920 925

Thr Met Leu Ile Pro Val Asn Gly Gly Lys Gly Leu Ala Ser Asn Gly

930 935 940

Asn Met Val Arg Ile Asp Val Phe Arg Ala Glu Glu Lys Gly Lys Lys

945 950 955 960

Lys Tyr Tyr Phe Ile Pro Val Tyr Val Ala Asp Thr Val Lys Glu Glu

965 970 975

Leu Pro Asn Arg Ala Val Leu Ala His Lys Pro Tyr Glu Ala Trp Lys

980 985 990

Ile Met Lys Glu Glu Asn Phe Ile Phe Ser Leu Tyr Pro Asn Asp Leu

995 1000 1005

Ile Phe Val Asp Ala Gly Lys Glu Ile Pro Phe Lys Ala Ala Leu

1010 1015 1020

Lys Gly Ser Thr Leu Asp Pro Glu Lys Lys Ala Ser Arg Phe Leu

1025 1030 1035

Met Tyr Tyr Lys Gly Ala Asp Ile Ala Thr Gly Ser Ile Ser Gly

1040 1045 1050

Val Asn His Asp Glu Thr Tyr Lys Ala Arg Gly Val Gly Ile Gln

1055 1060 1065

Ser Leu Arg Glu Ile Lys Lys Cys Cys Ile Asp Val Leu Gly Asn

1070 1075 1080

Ile Ser Phe Ala Ser Lys Glu Lys Arg Gln Thr Phe Arg

1085 1090 1095

<210> 18

<211> 1110

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_16-1 chimera

<400> 18

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Leu Thr Val Ala Leu Thr Arg Gln Ser Tyr

740 745 750

Ile Gln Arg Leu Asn Thr Leu Glu Ala Ser His Glu His Met Glu Lys

755 760 765

Leu Val Lys Glu Ala Asn Thr Pro Tyr Lys Glu Lys Lys Ser Leu Leu

770 775 780

Glu Lys Trp Val Ala Leu Gln Pro His Phe Ser Val Glu Glu Val Thr

785 790 795 800

Thr Gln Val Asp Gly Ile Leu Val Ser Phe Arg Ala Gly Lys Arg Val

805 810 815

Thr Thr Pro Ala Arg Arg Ala Val Tyr His Gly Gly Lys Arg Thr Ile

820 825 830

Val Gln Arg Gly Ile Gln Val Pro Arg Gly Ala Leu Thr Glu Asp Thr

835 840 845

Ile Tyr Gly Lys Leu Gly Asp Lys Phe Val Val Lys Tyr Ala Leu Asp

850 855 860

His Pro Ser Met Lys Pro Glu Asn Ile Val Asp Pro Thr Ile Arg Leu

865 870 875 880

Leu Val Glu Asn Arg Ile Thr Ala Leu Gly Lys Lys Asp Ala Phe Lys

885 890 895

Thr Pro Leu Tyr Ser Ala Glu Gly Met Glu Ile Lys Ser Val Arg Cys

900 905 910

Tyr Thr Ser Leu Ser Glu Lys Gly Val Val Pro Ile Lys Tyr Asn Glu

915 920 925

Lys Gly Asn Ala Ile Gly Phe Ala Lys Lys Gly Asn Asn His His Val

930 935 940

Ala Ile Tyr Lys Asp Gln Ser Gly Gln Tyr Gln Glu Met Val Val Ser

945 950 955 960

Phe Trp Asp Ala Val Glu Arg Lys Leu Tyr Gly Val Pro Thr Val Ile

965 970 975

Thr Asn Pro Lys Thr Val Trp Asp Glu Leu Leu Glu Lys Glu Leu Pro

980 985 990

Gln Asp Phe Leu Glu Lys Leu Pro Lys Asp Asn Trp Gln Tyr Val Leu

995 1000 1005

Ser Met Gln Glu Asn Glu Met Phe Val Leu Gly Met Glu Glu Asp

1010 1015 1020

Glu Phe Asn Asp Ala Ile Asp Thr Gln Asp Tyr Asn Thr Leu Asn

1025 1030 1035

Lys His Leu Tyr Arg Val Gln Lys Leu Ser His Ala Asp Tyr Thr

1040 1045 1050

Phe Arg Phe His Thr Glu Thr Lys Val Asp Asp Lys Tyr Asp Gly

1055 1060 1065

Val Glu Asn Gly Arg Asn Thr Ser Met Ser Leu Lys Ala Leu Val

1070 1075 1080

Arg Ile Arg Ser Phe Asn Gly Leu Phe Thr Gln Phe Pro His Lys

1085 1090 1095

Val Lys Ile Asp Ile Met Gly Arg Ile Thr Lys Ala

1100 1105 1110

<210> 19

<211> 1174

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_16-2 chimera

<400> 19

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Leu Val Val Ala Cys Thr Lys Gln Ser Tyr

740 745 750

Ile Gln Arg Leu Asn Asn Leu Asn Thr Glu Arg Asp Ala Met Tyr Gln

755 760 765

Asp Ile Glu Ala Gln Ser Val Glu Trp Lys Glu Lys His Ser Leu Leu

770 775 780

Glu Lys Trp Ile Lys Leu Gln Pro His Pro Thr Val Ser Glu Val Thr

785 790 795 800

Asp Lys Val Asp Glu Ile Leu Val Ser Phe Lys Ala Gly Lys Arg Val

805 810 815

Ala Thr Leu Gly Lys Arg Ser Val Tyr Lys Asn Gly Lys Lys Thr Val

820 825 830

Val Gln Asn Asn Ile Ile Val Pro Arg Gly Ala Leu Cys Glu Glu Ser

835 840 845

Val Tyr Gly Gln Ile Asn Leu Ile Glu Lys Asn Lys Pro Ile Lys Tyr

850 855 860

Leu Phe Glu Asn Pro Ser Leu Ile Phe Lys Pro Tyr Ile Lys Ala Leu

865 870 875 880

Val Glu Glu Arg Leu Lys Glu Tyr Asn Gly Asp Thr Ser Lys Ala Ile

885 890 895

Ser Ser Leu Lys Asn Asn Pro Ile Tyr Leu Arg Lys Asp Lys Ser Val

900 905 910

Val Leu Glu Tyr Gly Thr Cys Tyr Lys Lys Glu Tyr Val Lys Lys Tyr

915 920 925

Ser Leu Asn Ser Ile Lys Ala Lys Asp Val Asp Ser Ile Ile Asp Lys

930 935 940

His Ile Arg Glu Val Val Arg Gln Arg Leu Glu Asp Asn Asn Asn Asn

945 950 955 960

Glu Lys Ala Ala Phe Ala Ser Pro Leu Tyr Ala Asp Lys Gln Lys Gln

965 970 975

Ile Pro Ile Lys Ser Val Arg Cys Thr Thr Gly Ile Asn Ile Ala Ala

980 985 990

Pro Val Asn Tyr Asn Glu Ser Asn Asp Pro Ile Ser Phe Val Lys Pro

995 1000 1005

Gly Asn Asn His His Ile Ala Ile Tyr Lys Asp Lys Asp Gly Lys

1010 1015 1020

Arg Gln Glu His Ile Val Thr Phe Trp His Ala Val Glu Arg Lys

1025 1030 1035

Lys Tyr Gly Met Pro Val Val Ile Thr Asn Pro Lys Glu Ile Trp

1040 1045 1050

Asp Leu Ile Ile Glu Lys Ser Leu Asp Leu Pro Glu Ser Phe Leu

1055 1060 1065

Asn Cys Leu Pro Asn Ser Asp Trp Asn Tyr Glu Ile Ser Met Gln

1070 1075 1080

Gln Asn Glu Met Phe Val Met Gly Met Ser Glu Asp Glu Phe Gln

1085 1090 1095

Asp Ala Ile Arg Asn Asn Asp Tyr Lys Thr Leu Asn Lys Tyr Leu

1100 1105 1110

Tyr Arg Val Gln Ser Val Ser Glu Ser Asp Tyr Trp Leu Arg Leu

1115 1120 1125

His Ile Glu Thr Met Asn Asp Lys Thr Pro Glu Gly Asn Ile Ile

1130 1135 1140

Lys Lys Tyr Tyr Arg Ile Lys Ser Ile Asn Thr Phe Phe Asn Phe

1145 1150 1155

Asn Pro His Lys Val Lys Ile Thr Leu Leu Gly Glu Ile Gln Ser

1160 1165 1170

Ser

<210> 20

<211> 1094

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_18-1 chimera

<400> 20

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Tyr Leu Asn Ala Val Val Gly Asn Val Tyr

740 745 750

His Glu Lys Phe Thr Lys Asn Pro Leu Arg Phe Val Arg Ser Gly Gln

755 760 765

Glu Tyr Ser Leu Asn Leu Ser Ala Leu Phe Gln Asn Trp Asn Ile Tyr

770 775 780

Lys Gly Gly Arg Val Ile Trp Gln Lys Gly Glu Asp Gly Ser Leu Glu

785 790 795 800

Thr Val Arg Ala Arg Met Ala Lys Asn Asp Pro Met Val Thr Arg Tyr

805 810 815

Cys Thr Glu Gly Arg Gly Ala Leu Tyr Asp Leu Gln Pro Met Lys Lys

820 825 830

Ser Lys Gly Gln Leu Pro Leu Lys Ser Ser Asp Glu Arg Leu Gln His

835 840 845

Ile Asp Arg Tyr Gly Gly Tyr Asn Lys Leu Ala Gly Ala Tyr Phe Thr

850 855 860

Leu Ala Ala Tyr Tyr Lys Lys Gly Lys Arg Val Lys Ser Ile Glu Ser

865 870 875 880

Val Pro Leu Tyr Leu Ala Ala Lys Leu Gln Arg Asp Pro Ala Ala Leu

885 890 895

Gln Gln Tyr Leu Ala Asp Gln Leu Gly Thr Asp Arg Val Glu Ile Leu

900 905 910

Val Pro Glu Ile Lys Leu Gly Thr Leu Phe Lys Trp Asn Gly Tyr Pro

915 920 925

Met Thr Leu Ser Gly Arg Thr Gly Pro Gln Leu Leu Phe Arg Asn Ala

930 935 940

Ala Glu Leu Arg Thr Asn Ala Glu Gln Glu Gln Tyr Ile Lys Lys Met

945 950 955 960

Ser Arg Tyr Leu Glu Lys Cys Lys Gly Arg Lys Glu Pro Leu Pro Ile

965 970 975

Arg Pro Ala Tyr Asp Lys Leu Thr Pro Glu Glu Asn Leu Gln Leu Tyr

980 985 990

Asp Ala Phe Thr Gln Trp Leu Thr Ser Gly Ile Tyr Ala Lys Arg Leu

995 1000 1005

Ser Leu Gln Gly Lys Phe Leu Leu Glu Lys Arg Asp Ala Phe Ala

1010 1015 1020

Ala Leu Ser Pro Glu Ala Gln Val Arg Gln Leu Met Glu Ile Leu

1025 1030 1035

His Leu Phe Gln Cys Asn Pro Val Ala Ala Asn Leu Ser Glu Leu

1040 1045 1050

Gly Gly Ala Ala His Ala Gly Ile Leu Leu Ala Ser Lys Asn Ile

1055 1060 1065

Asp Gly Lys Val Pro Val Ser Ile Val His Gln Ser Val Thr Gly

1070 1075 1080

Tyr Phe Thr Gln Glu Val Cys Leu Asn Asp Leu

1085 1090

<210> 21

<211> 1101

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_21-1 chimera

<400> 21

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Ile Ala Cys Ile Thr Pro Gly Met

740 745 750

Ile Gln Lys Ile Thr Lys Tyr Ala Gln Asn His Glu Arg Phe Tyr Ala

755 760 765

Thr Ala Lys Gly Tyr Val Asp Ile Glu Thr Gly Glu Val Leu Thr Arg

770 775 780

Ser Glu Tyr Glu Ala Met Asp Asp Ile Arg Phe Pro Glu Pro Trp Pro

785 790 795 800

Gly Phe Arg Ser Glu Leu Glu Ala Arg Val Ser Glu His Pro Gln Glu

805 810 815

Ala Ile Ala Arg Leu Lys Leu Pro His Tyr Glu Asn Ser Glu Glu Ile

820 825 830

Arg Pro Ile Phe Val Ser Arg Met Pro Asn His Lys Val Thr Gly Ala

835 840 845

Ala His Leu Glu Thr Ile Arg Ser Lys Lys Gly Gly Ala Gly Ser Thr

850 855 860

Val Thr Lys Thr Ala Leu Pro Asp Leu Lys Leu Asp Lys Asn Gly Glu

865 870 875 880

Ile Ala Gly Tyr Tyr Arg Lys Glu Asp Asp Pro Leu Leu Tyr Glu Ala

885 890 895

Leu Lys Ala Arg Leu Lys Ala Phe Gly Gly Asp Gly Lys Lys Ala Phe

900 905 910

Ala Glu Pro Phe His Lys Pro Lys His Asn Gly Glu Pro Gly Pro Ile

915 920 925

Val Lys Lys Val Lys Ile Gln Glu Ser Ala Thr Leu Thr Val Pro Val

930 935 940

Asn His Gly Ile Ala Ala Asn Gly Ser Met Val Arg Leu Asp Val Phe

945 950 955 960

His Val Asp Gly Asp Gly Tyr Tyr Phe Val Pro Ile Tyr Thr Ser Asp

965 970 975

Thr Val Lys Pro Glu Leu Pro Asn Arg Ala Val Val Ala Gly Arg Arg

980 985 990

Val Gln Glu Trp Lys Val Met Asp Asp Ser Tyr Phe Lys Phe Ser Leu

995 1000 1005

Tyr Pro Lys Asp Leu Ile Arg Ile Arg Ser Lys Lys Gly Ile Lys

1010 1015 1020

Leu Lys Ala Val Asn Arg Asn Ala Asp Leu Gln Glu Tyr Ser Thr

1025 1030 1035

Asn Asp Cys Leu Cys Tyr Phe Val Lys Phe Asn Ile Ser Thr Gly

1040 1045 1050

Ala Leu Ser Val Glu Asn His Asp Arg Lys Phe Glu Gln Pro Gly

1055 1060 1065

Leu Gly Gly Lys Thr Leu Leu Ser Ile Glu Lys Tyr Gln Val Asp

1070 1075 1080

Val Leu Gly Asn Tyr Ser Pro Val Ala Leu Pro Glu Lys Arg Met

1085 1090 1095

Lys Phe Arg

1100

<210> 22

<211> 1172

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_22-2 chimera

<400> 22

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ile Ala Ile Ala Cys Ile Asn Arg Ser Ile

740 745 750

Val Asn Tyr Leu Asn Asn Ala Ala Ala Asn Gln Thr Glu Arg Glu Asp

755 760 765

Leu Arg Arg Ala Val Cys Ile Pro Glu Arg Asn Gly Gln Thr Lys Arg

770 775 780

Gln Leu Arg Ser Pro Trp His Cys Phe Ala Arg Asp Ala Glu Asn Ala

785 790 795 800

Leu Arg Gln Ile Val Val Ser Phe Lys Gln Asn Leu Arg Val Ala Thr

805 810 815

Lys Ala Thr Asn Ser Tyr Glu Cys Phe Asp Thr Ala Ser Gly Lys Lys

820 825 830

Ile Arg Lys His Gln Ser Asn Arg Glu His Tyr Ala Ile Arg Lys Pro

835 840 845

Leu His Lys Asp Ser Val Tyr Gly Glu Val Ile Leu Thr Ser Ile Ala

850 855 860

Ser Val Asn Leu Lys Lys Ala Leu Leu Lys Ala Glu Arg Ile Leu Asp

865 870 875 880

Lys Arg Leu Lys Glu Lys Ile Phe Glu Leu Arg Lys Leu Tyr Asn Tyr

885 890 895

Ser Asn Lys Gln Ile Glu Glu His Leu Thr Lys Val Cys Ile Asn Cys

900 905 910

Pro Glu Trp Lys Asn Tyr Asp Phe Lys Lys Ile Ala Val Arg Ile Leu

915 920 925

Ser Asn Asp Ala Asp Ala Thr His Ile Val Ala Ile Arg Lys Pro Leu

930 935 940

Asp Glu Ser Phe Asp Glu Val Lys Ile Asn Thr Ile Thr Asp Thr Gly

945 950 955 960

Ile Gln Lys Ile Leu Leu Asn His Leu Ser Arg Tyr Ala Asp Asp Pro

965 970 975

Lys Lys Ala Phe Ser Pro Glu Gly Ile Glu Asp Met Asn Ala Asn Ile

980 985 990

Ala Ser Leu Asn Gly Gly Lys Gln His Leu Pro Ile Tyr Lys Val Arg

995 1000 1005

Val Ser Glu Lys Asp Asn Gly Gly Tyr Phe Pro Ile Gly Gln Lys

1010 1015 1020

Gly Asn Arg Pro Lys Lys Tyr Val Thr Thr Ala Lys Asp Thr Asn

1025 1030 1035

Leu Phe Phe Ala Val Tyr Ala Asp Ser Lys Gly Lys Arg Ser Tyr

1040 1045 1050

Lys Thr Ile Asp Leu Arg Thr Ala Ile Glu Cys Arg Lys Gln Gly

1055 1060 1065

Leu Ser Val Ala Pro Ser Ile Asn Glu Lys Gly Asp Lys Leu Leu

1070 1075 1080

Phe Thr Leu Ser Pro Asn Asp Leu Val Tyr Met Pro Ser Glu Gly

1085 1090 1095

Glu Glu Ala Asn Gly Phe Ala Ile Asp Asn Asn Leu Asn Lys Asp

1100 1105 1110

Gln Ile Tyr Lys Met Val Ser Ala Asn Asn Lys Gln Cys Phe Phe

1115 1120 1125

Ile Pro His Thr Val Ala Asp Phe Ile Ser Arg Gly Glu Glu Tyr

1130 1135 1140

Asn Ser His Asn Lys Ile Glu Leu Thr Glu Asp Arg Arg Ser Ile

1145 1150 1155

Lys Glu His Cys Val Pro Leu Lys Val Asn Arg Leu Gly Lys

1160 1165 1170

<210> 23

<211> 1112

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_23-1 chimera

<400> 23

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Thr Tyr

740 745 750

Ser Thr Lys Phe Thr Asn Asn Pro Leu Asn Phe Ile Lys Ala Gly Ala

755 760 765

Lys Arg Pro Gln Asp Asn Gln Phe Lys Tyr Asn Met Asp Lys Ile Phe

770 775 780

Asp Tyr Asn Val Ile Ser Arg Gly Glu Arg Ala Trp Ile Ala Gly Ser

785 790 795 800

Asp Gly Ser Ile Cys Thr Val Lys Lys Phe Met Ser Arg Asn Thr Val

805 810 815

Leu Ile Thr Arg Lys Ala Lys Glu Val His Gly Ala Leu Ser Asn Lys

820 825 830

Ala Thr Ile Trp Gly Lys Asn Val Ala Lys Pro Gly Ala Tyr Leu Pro

835 840 845

Val Lys Ser Thr Asp Leu Lys Ala Gln Asp Val Thr Lys Tyr Gly Gly

850 855 860

Ile Thr Ser Ile Ala Asn Ser Gly Tyr Thr Leu Ala Glu Tyr Lys Val

865 870 875 880

Asn Gly Lys Thr Thr Arg Ser Leu Glu Ala Leu Pro Val Tyr Leu Gly

885 890 895

Arg Ala Glu Gln Leu Thr Glu Lys Thr Val Val Asp Tyr Leu Ser Ser

900 905 910

Ser Leu Gln Glu Ser Ser Lys Lys Lys Ile Glu Asp Ile Gln Val Arg

915 920 925

Lys Leu Phe Ile Pro Gln Gly Ser Lys Val Lys Ile Asp Gly Phe Cys

930 935 940

Tyr Tyr Leu Gly Gly Lys Thr Gly Asp Ser Ile Tyr Leu Asn Asn Ala

945 950 955 960

Val Pro Leu Tyr Leu Ser Ser Thr Ser Glu Glu Tyr Leu Arg Lys Leu

965 970 975

Leu Lys Ala Val Glu Asn Asn Asn Tyr Asn Glu Arg Asp Lys Asn Gly

980 985 990

Gln Ile Ile Leu Thr Ala Pro Lys Asn Val Gln Leu Leu Ser Ser Ile

995 1000 1005

Phe Asp Lys Leu Arg Ser Lys Pro Phe Ser Asn Asn Lys Trp Asn

1010 1015 1020

Ile Tyr Phe Ser Ile Val Asn Gly Lys Glu Thr Lys Val Glu Gln

1025 1030 1035

Leu Phe Ser Lys Leu Ser Ile Asp Lys Gln Ala Glu Val Ile Ser

1040 1045 1050

Gln Ile Val Ile Trp Ile Asn Ser Ser Arg Gln Asn Val Asn Leu

1055 1060 1065

Ser Leu Ile Gly Gly Ser Ala His Ser Gly Thr Gln Ala Leu Ser

1070 1075 1080

Lys Thr Val Ser Arg Leu Asn Glu Cys Met Leu Ile Ser Gln Ser

1085 1090 1095

Ile Thr Gly Ile Tyr Glu His Ser Val Asp Leu Leu Thr Ile

1100 1105 1110

<210> 24

<211> 1090

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_SaCas9 chimeras

<400> 24

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe

740 745 750

Lys Glu Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln

755 760 765

Met Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu

770 775 780

Gln Glu Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile

785 790 795 800

Lys Asp Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro

805 810 815

Asn Arg Glu Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp

820 825 830

Lys Gly Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys

835 840 845

Asp Asn Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu

850 855 860

Leu Met Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile

865 870 875 880

Met Glu Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu

885 890 895

Glu Thr Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro

900 905 910

Val Ile Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu

915 920 925

Asp Ile Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu

930 935 940

Ser Leu Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr

945 950 955 960

Lys Phe Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr

965 970 975

Tyr Glu Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys

980 985 990

Ile Ser Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu

995 1000 1005

Ile Lys Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn

1010 1015 1020

Asp Leu Leu Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr

1025 1030 1035

Arg Glu Tyr Leu Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile

1040 1045 1050

Ile Lys Thr Ile Ala Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser

1055 1060 1065

Thr Asp Ile Leu Gly Asn Leu Tyr Glu Val Lys Ser Lys Lys His

1070 1075 1080

Pro Gln Ile Ile Lys Lys Gly

1085 1090

<210> 25

<211> 1123

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_SpCas9 chimeras

<400> 25

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu

740 745 750

Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr

755 760 765

Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile

770 775 780

Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe

785 790 795 800

Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro

805 810 815

Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly

820 825 830

Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn

835 840 845

Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser

850 855 860

Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp

865 870 875 880

Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr

885 890 895

Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

900 905 910

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

915 920 925

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu

930 935 940

Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu

945 950 955 960

Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln

965 970 975

Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr

980 985 990

Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu

995 1000 1005

Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu

1010 1015 1020

Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala

1025 1030 1035

Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg

1040 1045 1050

Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1055 1060 1065

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1070 1075 1080

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu

1085 1090 1095

Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr

1100 1105 1110

Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1115 1120

<210> 26

<211> 1105

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_15-1-WP chimera

<400> 26

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Met Thr Leu Leu Asn Arg Ser Val

740 745 750

Ala Leu Thr Leu Glu Gln Arg Ser Gln Leu Arg Arg Thr Phe Tyr Glu

755 760 765

Gln Gly Leu Asp Lys Leu Asp Arg Asn Gln Leu Lys Pro Glu Glu Asp

770 775 780

Trp Arg Asp Phe Thr Gly Leu Ala Pro Ala Ser Gln Glu Lys Phe Leu

785 790 795 800

Glu Trp Arg Lys Ala Ala Thr Ile Leu Gly Asp Leu Leu Ala Glu Ala

805 810 815

Ile Glu Asp Asp Ser Ile Ala Val Val Ser Pro Leu Arg Leu Arg Pro

820 825 830

Gln Asn Gly Ser Val His Leu Glu Lys Ile His Gly Pro Ala His Lys

835 840 845

Glu Thr Ile Arg Ser Gly Arg Leu Lys Glu Glu Gly Tyr Thr Ile Ser

850 855 860

Lys Thr Ala Leu Ile Asp Leu Lys Leu Thr Glu Asp Lys Glu Glu Ile

865 870 875 880

Lys Gly Tyr Tyr Asn Lys Glu Ser Asp Arg Leu Leu Tyr Glu Ala Leu

885 890 895

Lys Lys Gln Leu Gln Arg Tyr Gly Gly Lys Ala Lys Glu Ala Phe Lys

900 905 910

Glu Pro Phe His Lys Pro Lys Ala Asp Gly Thr Pro Gly Pro Ile Val

915 920 925

Asn Lys Val Lys Ile Met Glu Lys Ser Thr Met Leu Ile Pro Val Asn

930 935 940

Gly Gly Lys Gly Leu Ala Ser Asn Gly Asn Met Val Arg Ile Asp Val

945 950 955 960

Phe Arg Ala Glu Glu Lys Gly Lys Lys Lys Tyr Tyr Phe Ile Pro Val

965 970 975

Tyr Val Ala Asp Thr Val Lys Glu Glu Leu Pro Asn Arg Ala Val Leu

980 985 990

Ala His Lys Pro Tyr Glu Ala Trp Lys Ile Met Lys Glu Glu Asn Phe

995 1000 1005

Ile Phe Ser Leu Tyr Pro Asn Asp Leu Ile Phe Val Asp Ala Gly

1010 1015 1020

Lys Glu Ile Pro Phe Lys Ala Ala Leu Lys Gly Ser Thr Leu Asp

1025 1030 1035

Pro Glu Lys Lys Ala Ser Arg Phe Leu Met Tyr Tyr Lys Gly Ala

1040 1045 1050

Asp Ile Ala Thr Gly Ser Ile Ser Gly Val Asn His Asp Glu Thr

1055 1060 1065

Tyr Lys Ala Arg Gly Val Gly Ile Gln Ser Leu Arg Glu Ile Lys

1070 1075 1080

Lys Cys Cys Ile Asp Val Leu Gly Asn Ile Ser Phe Ala Ser Lys

1085 1090 1095

Glu Lys Arg Gln Thr Phe Arg

1100 1105

<210> 27

<211> 1074

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3 chimeric effector

<220>

<223> description: MG3-6_15-1-P chimeras

<400> 27

Met Ser Thr Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp

1 5 10 15

Arg Ser Val Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro

20 25 30

Ile Gln Lys Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp

35 40 45

Pro Thr Lys Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile

50 55 60

Ala Arg Arg Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg

65 70 75 80

Asn Leu Asp Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly

85 90 95

Pro Glu Pro Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala

100 105 110

Ser Ile Lys Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg

115 120 125

Ala Val Arg His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp

130 135 140

Ser Leu Asp Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe

145 150 155 160

Glu Ile Ile Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro

165 170 175

Ala Asn Pro Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu

180 185 190

Val Leu Leu Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val

195 200 205

Arg Gly Thr Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu

210 215 220

Ala Glu Leu Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr

225 230 235 240

Glu Ala Leu Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro

245 250 255

Lys Glu Arg Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr

260 265 270

Ile Arg Ala Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val

275 280 285

Ala Asn Leu Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr

290 295 300

Glu Ala Glu Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp

305 310 315 320

Lys Glu Gln Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro

325 330 335

Gly Asn Arg Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr

340 345 350

Ala Pro Tyr Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys

355 360 365

Lys Thr Glu Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu

370 375 380

Arg Ser Leu Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu

385 390 395 400

Glu Ala Ala Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro

405 410 415

Ala Glu Glu Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg

420 425 430

Val Ala Tyr Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His

435 440 445

Glu Tyr Arg Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val

450 455 460

Asp Asp Thr Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly

465 470 475 480

Gln Pro Ala Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu

485 490 495

Asp Cys Glu Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His

500 505 510

Thr Arg Thr Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn

515 520 525

Glu Gln Lys Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu

530 535 540

Arg Glu Ser Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His

545 550 555 560

Leu Ile Val Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met

565 570 575

Ile Thr Thr Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly

580 585 590

Gly Gly Ser Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys

595 600 605

Asn Ala Lys Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val

610 615 620

Lys Ser Gln Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys

625 630 635 640

Asp Ser Lys Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn

645 650 655

Gln Thr Glu Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr

660 665 670

Ser Tyr Ala Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn

675 680 685

Glu Gly Leu Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr

690 695 700

Ala Gly Ala Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu

705 710 715 720

Arg Ile Leu Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg

725 730 735

His His Ala Val Asp Ala Ala Val Met Thr Leu Leu Asn Arg Ser Val

740 745 750

Ala Leu Thr Leu Glu Gln Arg Ser Gln Leu Arg Arg Thr Phe Tyr Glu

755 760 765

Gln Gly Leu Asp Lys Leu Asp Arg Asn Gln Leu Lys Pro Glu Glu Asp

770 775 780

Trp Arg Asp Phe Thr Gly Leu Ala Pro Ala Ser Gln Glu Lys Phe Leu

785 790 795 800

Glu Trp Arg Lys Ala Ala Thr Ile Leu Gly Asp Leu Leu Ala Glu Ala

805 810 815

Ile Glu Asp Asp Ser Ile Ala Val Val Ser Pro Leu Arg Leu Arg Pro

820 825 830

Gln Asn Gly Ser Val His Leu Glu Thr Ile Ser Ala Val Lys Lys Gln

835 840 845

Thr Leu Gly Ser Asp Trp Pro Ala Asp Ala Val Lys Arg Ile Val Asp

850 855 860

Pro Glu Ile Tyr Leu Ala Met Lys Asp Ala Leu Gly Lys Leu Lys Glu

865 870 875 880

Leu Pro Glu Asp Ser Ala Arg Ser Leu Glu Leu Pro Asp Gly Arg Phe

885 890 895

Val Glu Ala Asp Asp Glu Val Leu Phe Phe Pro Glu Asn Ala Ala Ser

900 905 910

Ile Leu Thr Pro Arg Gly Val Ala Glu Ile Asn Met Val Arg Ile Asp

915 920 925

Val Phe Arg Ala Glu Glu Lys Gly Lys Lys Lys Tyr Tyr Phe Ile Pro

930 935 940

Val Tyr Val Ala Asp Thr Val Lys Glu Glu Leu Pro Asn Arg Ala Val

945 950 955 960

Leu Ala His Lys Pro Tyr Glu Ala Trp Lys Ile Met Lys Glu Glu Asn

965 970 975

Phe Ile Phe Ser Leu Tyr Pro Asn Asp Leu Ile Phe Val Asp Ala Gly

980 985 990

Lys Glu Ile Pro Phe Lys Ala Ala Leu Lys Gly Ser Thr Leu Asp Pro

995 1000 1005

Glu Lys Lys Ala Ser Arg Phe Leu Met Tyr Tyr Lys Gly Ala Asp

1010 1015 1020

Ile Ala Thr Gly Ser Ile Ser Gly Val Asn His Asp Glu Thr Tyr

1025 1030 1035

Lys Ala Arg Gly Val Gly Ile Gln Ser Leu Arg Glu Ile Lys Lys

1040 1045 1050

Cys Cys Ile Asp Val Leu Gly Asn Ile Ser Phe Ala Ser Lys Glu

1055 1060 1065

Lys Arg Gln Thr Phe Arg

1070

<210> 28

<211> 121

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG1 sgRNA

<220>

<223> description: MG1-4 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 28

nnnnnnnnnn nnnnnnnnnn nngttttgat ttactcgaaa gagtccaatc ataattgacc 60

ggagaataat tgattcctct acaatgtacg aataaattca ttctctaaac cttaaaaatt 120

t 121

<210> 29

<211> 99

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 sgRNA

<220>

<223> description: MG1-5 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 29

nnnnnnnnnn nnnnnnnnnn nngttttgac ttgaaaaagt cttaactgat tttgccgaat 60

tttaagctct acgtagtacc ttggaattcg gcatatttt 99

<210> 30

<211> 99

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 sgRNA

<220>

<223> description: MG1-6 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 30

nnnnnnnnnn nnnnnnnnnn nngttttgac ttgaaaaagt cttaactgat tttgccgaat 60

ttcaagctct gcattgcacc ttggcattcg gcatatttt 99

<210> 31

<211> 99

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 sgRNA

<220>

<223> description: MG1-7 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 31

nnnnnnnnnn nnnnnnnnnn nngttttgac tttgaaaaaa gtcttaactg attttgccga 60

attttaagct ctgcatggca ccttgaaatt cggcatttt 99

<210> 32

<211> 100

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG2 sgRNA

<220>

<223> description: MG2-7 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 32

nnnnnnnnnn nnnnnnnnnn nngctttgcc ttggaaacaa gacaaagtta attaaggcag 60

ttcggacctc tactttgtac gtcaggatag aaagcctttt 100

<210> 33

<211> 115

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 sgRNA

<220>

<223> description: MG3-3 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 33

nnnnnnnnnn nnnnnnnnnn nngttgggaa tcttgaaaaa gattcccaat aaggcacatt 60

tttagtgctg acttctcacc gtccagggtt cattgaacaa tgggcggtat gtttt 115

<210> 34

<211> 126

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 sgRNA

<220>

<223> description: MG3-4 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 34

nnnnnnnnnn nnnnnnnnnn nngttgagaa tctttcatta gaaataacga aagattctta 60

ataaggcgtc cttccgatgc tgacttctca ccgtccgttt tccaatagga gcgggcggta 120

tgtttt 126

<210> 35

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 sgRNA

<220>

<223> description: MG3-6 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 35

nnnnnnnnnn nnnnnnnnnn nngttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 36

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 sgRNA

<220>

<223> description: MG3-7 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 36

nnnnnnnnnn nnnnnnnnnn nngttgggaa ccgaaaggtt cccaataagg cgcatcttgg 60

cgctgacttc tcaccgtcct cttgctgctt agcagagggc ggtatgtttt 110

<210> 37

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 sgRNA

<220>

<223> description: MG3-8 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 37

nnnnnnnnnn nnnnnnnnnn nngttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc ggctcctctt aggaacgggc ggtatgtttt 110

<210> 38

<211> 124

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG4 sgRNA

<220>

<223> description: MG4-5 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 38

nnnnnnnnnn nnnnnnnnnn nngctgtggc ttgcggggga aaccccttgt cacagtaagg 60

gactttcgtt cgcgaaaggc aacctcgcca gcatcgctgg gcgaggacca gggcaaggcg 120

attt 124

<210> 39

<211> 118

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG14 sgRNA

<220>

<223> description: MG14-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 39

nnnnnnnnnn nnnnnnnnnn nngtcttgag cgaaagctcc agacaagggg agccacttaa 60

gtggcttacc cgtaaagtaa cccccgttca atcttcggat tgggcggggc gaactttt 118

<210> 40

<211> 118

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG15 sgRNA

<220>

<223> description: MG15-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 40

nnnnnnnnnn nnnnnnnnnn nngttgtaat tccctagaaa taggttatta caataaggtc 60

caacaggagt gttggtaccg taaagctcta acggcaccca cgggtgccgt tatctttt 118

<210> 41

<211> 162

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG16 sgRNA

<220>

<223> description: MG16-2 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 41

nnnnnnnnnn nnnnnnnnnn nngttgtgaa ttgctttaaa ttgaaaaatt taagcaattc 60

acaataagga ttattccgtt gtgaaaacat ttaaagcggg gtcaacagcc tcgctttctt 120

tttgagtcta tgagacatta ggtcaataag tctatgagtt tt 162

<210> 42

<211> 101

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG18 sgRNA

<220>

<223> description: MG18-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 42

nnnnnnnnnn nnnnnnnnnn nngtttgaga gtagtgaaaa ctacgagttc aaatacaatt 60

ttttcaaatt gccctatagg gccctcacag tgtgagattt t 101

<210> 43

<211> 117

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG21 sgRNA

<220>

<223> description: MG21-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 43

nnnnnnnnnn nnnnnnnnnn nngttgtagt tccccttttg aaaaaaagtg tgttactgca 60

ataaggtaaa acaccacgaa gctctgccct aactgcctta gcagttaggg catcttt 117

<210> 44

<211> 134

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG22 sgRNA

<220>

<223> description: MG22-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 44

nnnnnnnnnn nnnnnnnnnn nngttgtgaa ttgctttcaa attagaaata attgaaagca 60

attcgcaata aggattattc cgttgtgaaa acatttcgag tggcttcgtg aaattcacga 120

agtcacttcg tttt 134

<210> 45

<211> 113

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG23 sgRNA

<220>

<223> description: MG23-1 sgRNA

<220>

<221> modified_base

<222> (1)..(22)

<223> a, c, t, g, unknown or other

<400> 45

nnnnnnnnnn nnnnnnnnnn nngtttgaga acctgaaaag gtgagtgcaa ataaggttta 60

accgaaattg tttacctgca ttgtgcagta taagaaagac cgcgaggtct ttt 113

<210> 46

<211> 4

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 PAM

<220>

<223> description: MG1-4 PAM

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, t, g, unknown or other

<400> 46

nrrr 4

<210> 47

<211> 6

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 PAM

<220>

<223> description: MG1-5 PAM

<220>

<221> modified_base

<222> (1)..(4)

<223> a, c, t, g, unknown or other

<400> 47

nnnnyy 6

<210> 48

<211> 6

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 PAM

<220>

<223> description: MG1-6 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<400> 48

nnrray 6

<210> 49

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG1 PAM

<220>

<223> description: MG1-7 PAM

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, t, g, unknown or other

<400> 49

nrrraag 7

<210> 50

<211> 6

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG2 PAM

<220>

<223> description: MG2-7 PAM

<220>

<221> modified_base

<222> (1)..(3)

<223> a, c, t, g, unknown or other

<400> 50

nnnrta 6

<210> 51

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 PAM

<220>

<223> description: MG3-3 PAM

<220>

<221> modified_base

<222> (1)..(3)

<223> a, c, t, g, unknown or other

<400> 51

nnncccyr 8

<210> 52

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 PAM

<220>

<223> description: MG3-4 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (8)..(8)

<223> a, c, t, g, unknown or other

<400> 52

nnaaaabn 8

<210> 53

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 PAM

<220>

<223> description: MG3-6 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (6)..(6)

<223> a, c, t, g, unknown or other

<400> 53

nnrggnt 7

<210> 54

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 PAM

<220>

<223> description: MG3-7 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, t, g, unknown or other

<400> 54

nnrnyay 7

<210> 55

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 PAM

<220>

<223> description: MG3-8 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<400> 55

nnrggty 7

<210> 56

<211> 5

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG4 PAM

<220>

<223> description: MG4-5 PAM

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, t, g, unknown or other

<400> 56

nrccv 5

<210> 57

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG14 PAM

<220>

<223> description: MG14-1 PAM

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (3)..(4)

<223> a, c, t, g, unknown or other

<400> 57

nrnngrka 8

<210> 58

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG15 PAM

<220>

<223> description: MG15-1 PAM

<220>

<221> modified_base

<222> (2)..(4)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (6)..(6)

<223> a, c, t, g, unknown or other

<400> 58

cnnncnaa 8

<210> 59

<211> 6

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG18 PAM

<220>

<223> description: MG18-1 PAM

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, t, g, unknown or other

<400> 59

nrwart 6

<210> 60

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_3-2 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<400> 60

nnrmyymw 8

<210> 61

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_3-3 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<400> 61

nnhcccyr 8

<210> 62

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_3-4 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (7)..(8)

<223> a, c, t, g, unknown or other

<400> 62

nnaaaann 8

<210> 63

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_3-7 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, t, g, unknown or other

<400> 63

nnrnyay 7

<210> 64

<211> 7

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_3-8 PAM

<220>

<221> modified_base

<222> (1)..(2)

<223> a, c, t, g, unknown or other

<400> 64

nnrggty 7

<210> 65

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG 3-6_15-1-WPPAM

<220>

<221> modified_base

<222> (2)..(4)

<223> a, c, t, g, unknown or other

<400> 65

cnnncwaa 8

<210> 66

<211> 8

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG3 chimeric PAM

<220>

<223> description: MG3-6_15-1-P PAM

<220>

<221> modified_base

<222> (1)..(4)

<223> a, c, t, g, unknown or other

<400> 66

nnnncwaa 8

<210> 67

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 45 for targeting albumin intron 1

<400> 67

tgccagttcc cgatcgttac aggttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 68

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 78 for targeting albumin intron 1

<400> 68

aaataccagg cttccattac tagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 69

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-6 guide sequence 24 for targeting albumin intron 1

<400> 69

atttacaaac atgacagaaa cagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 70

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 34 for targeting albumin intron 1

<400> 70

cttaggtcag tgaagagaag aagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 71

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 44 for targeting albumin intron 1

<400> 71

atgccagttc ccgatcgtta cagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 72

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 87 for targeting albumin intron 1

<400> 72

cttctcggcg aaacacaccc ctgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 73

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 81 for targeting albumin intron 1

<400> 73

ctagaaaaat acaagcagag atgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 74

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 72 for targeting albumin intron 1

<400> 74

aataataatc tagaaatcag cagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 75

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 16 for targeting albumin intron 1

<400> 75

ctgcctgctc gaccatgcta tagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 76

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 59 for targeting albumin intron 1

<400> 76

aggcaggccc tatgagaccg tagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 77

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 30 for targeting albumin intron 1

<400> 77

ttttaaaaat aataatgttg gtgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 78

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 20 for targeting albumin intron 1

<400> 78

tcgaccatgc tatactaaaa atgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 79

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 84 for targeting albumin intron 1

<400> 79

atacaagcag agatgaaaaa acgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 80

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 33 for targeting albumin intron 1

<400> 80

gcttaggtca gtgaagagaa gagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 81

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 64 for targeting albumin intron 1

<400> 81

aaagaaattt aaagctaagc ttgttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 82

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 53 for targeting albumin intron 1

<400> 82

ctaagacaat ggtaaataag aagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 83

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 73 for targeting albumin intron 1

<400> 83

taatctagaa atcagcacta aagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 84

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 74 for targeting albumin intron 1

<400> 84

aatctagaaa tcagcactaa aggttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 85

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 13 for targeting albumin intron 1

<400> 85

ctttaaattt cttttaatta aagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 86

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3 Hepa1-6 targeting guide (contained in sgRNA)

<220>

<223> description: MG3-6_3-4 guide sequence 19 for targeting albumin intron 1

<400> 86

ctcgaccatg ctatactaaa aagttgagaa tcgaaagatt cttaataagg catccttccg 60

atgctgactt ctcaccgtcc gttttccaat aggagcgggc ggtatgtttt 110

<210> 87

<211> 50

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: amplification of MG3-6 (718-840) against MG3-6 (1-840) _MG15-1 (818-1082)

<400> 87

gtcacccgcg aaagtcgtcg cgccggcggc atcgatgaac gcatcctgtt 50

<210> 88

<211> 54

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<400> 88

tagtctcttt gtgggcagga ccgtggatct tctctaagtg aacagatcca tttt 54

<210> 89

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: amplification of MG15-1 (818-1082) against MG3-6 (1-840) _MG15-1 (818-1082)

<400> 89

cgcccgcaaa atggatctgt tcacttagag aagatccacg gtcctgcc 48

<210> 90

<211> 50

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<400> 90

gttatcagtg gtggtggtgg tggtgctcga gacggaacgt ttggcgcttc 50

<210> 91

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: amplification of MG3-6 (718-922) against MG3-6 (1-922) _MG15-1 (931-1082)

<400> 91

ggcgcgaaag acgtcgatgc gaaccatgtt gatttccgct acgccacg 48

<210> 92

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: MG15-1 (931-1082) was amplified against MG3-6 (1-922) _MG15-1 (931-1082)

<400> 92

tcctgacccc gcgtggcgta gcggaaatca acatggttcg catcgacg 48

<210> 93

<211> 38

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: LA065 primers for PCR in PAM enrichment assays

<220>

<221> modified_base

<222> (14)..(18)

<223> a, c, t, g, unknown or other

<400> 93

gctcttccga tctnnnnnat tgacggcggc atcggact 38

<210> 94

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: LA125 primers for PCR in PAM enrichment assays

<220>

<221> modified_base

<222> (14)..(18)

<223> a, c, t, g, unknown or other

<400> 94

gctcttccga tctnnnnngg tgttggcggg tgtcg 35

<210> 95

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: LA003 PAM enriched adaptors (heteroduplex of LA003/LA 011)

<400> 95

attgacggcg gcatcggact 20

<210> 96

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: LA011 PAM enrichment adapter (heteroduplex LA003/LA 011)

<400> 96

agtccgatgc cgccgtcaat t 21

<210> 97

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 57F primer for Mulberry sequencing of edited Hepa1-6 cells

<400> 97

tctggcaaaa tgaagtgggt 20

<210> 98

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 1072R primer for Mulberry sequencing of edited Hepa1-6 cells

<400> 98

tgccacattg ctcagcacag 20

<210> 99

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 132F primer for Mulberry sequencing of edited Hepa1-6 cells

<400> 99

cgccgagaag cacgtaagag 20

<210> 100

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 282F primer for Mulberry sequencing of edited Hepa1-6 cells

<400> 100

ttgcatctga gaacccttag g 21

<210> 101

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 446R primers for Mulberry sequencing of edited Hepa1-6 cells

<400> 101

ccgtaataaa ttcaactgta tcc 23

<210> 102

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: 460F primer for Mulberry sequencing of edited Hepa1-6 cells

<400> 102

gcctgctcga ccatgctata 20

<210> 103

<211> 8755

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: plasmid(s)

<220>

<223> description: pMGX3-6 plasmid

<400> 103

ctcgagcacc accaccacca ccactgataa caaagcccga aaggaagctg agttggctgc 60

tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 120

ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc 180

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 240

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 300

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 360

gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 420

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 480

ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 540

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 600

atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 660

tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 720

atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 780

attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 840

gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 900

agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 960

aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 1020

cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 1080

cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 1140

actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 1200

cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 1260

ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 1320

ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 1380

gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 1440

gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1500

ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 1560

cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1620

caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 1680

taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 1740

cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 1800

cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 1860

gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1920

aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1980

cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2040

tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2100

acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2160

ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2220

ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2280

tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2340

tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2400

ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2460

gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2520

cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg 2580

catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg 2640

ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 2700

ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2760

ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2820

accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 2880

gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 2940

gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc 3000

cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct 3060

cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca 3120

actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt 3180

cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg 3240

gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc 3300

gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt 3360

tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg 3420

ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat 3480

ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 3540

gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 3600

gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 3660

gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 3720

tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 3780

tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3840

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 3900

tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 3960

agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 4020

tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 4080

tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 4140

gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 4200

gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 4260

tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 4320

ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 4380

gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 4440

gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 4500

gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 4560

tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 4620

cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 4680

tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 4740

tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 4800

tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 4860

cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 4920

atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 4980

ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc 5040

gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 5100

gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc 5160

ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg 5220

gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca 5280

ctataggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 5340

aaggagatat accatgtcaa ccgatatgaa gaattatcgc attggcgtgg acgtcgggga 5400

tcgcagcgta ggcttggcag cgattgagtt cgacgacgac gggctgccga ttcagaaact 5460

ggcattagtt acctttcgtc atgacggtgg cctcgaccca accaagaaca aaacgcctat 5520

gtcgcgcaaa gagacccgcg ggatcgcgcg ccgcaccatg cgtatgaatc gcgaacgcaa 5580

acgccgcctt cgcaatttgg acaacgtact ggagaactta ggctatagcg tacctgaggg 5640

acctgaaccg gaaacttacg aagcctggac gagccgcgca ctcttggcaa gcattaaatt 5700

agcatccgcc gacgaattaa acgaacacct ggttcgtgcc gttcgccaca tggcgcgcca 5760

tcgcggctgg gcaaacccct ggtggagcct ggatcaactc gaaaaggcat cacaagaacc 5820

cagcgaaacc ttcgaaatca tcttagcacg cgcacgcgaa ttattcggcg agaaagtacc 5880

agcgaacccc acccttggga tgctgggagc gctggcggca aacaacgaag tgctgctccg 5940

cccacgtgac gagaagaaac gcaaaacggg atacgtacgt ggaacgccgt tgatgttcgc 6000

acaagtgcgc caaggcgatc aattagccga attacgccgc atctgtgaag tacaaggcat 6060

cgaagatcaa tatgaagcgc tgcgtctggg tgtctttgat cacaaacatc cgtacgtccc 6120

aaaagagcgc gttggcaaag atccgttgaa cccgagcacc aaccgtacca tccgcgcaag 6180

ccttgaattc caagaatttc gtatcttaga cagtgttgcc aatctccgcg ttcgtatcgg 6240

cagtcgcgcc aaacgcgaac tcaccgaagc ggaatacgac gcagcggtag aatttctgat 6300

ggattacgcg gacaaagagc agcctagttg ggcggacgtg gcggaaaaga tcggggtgcc 6360

gggcaatcgt ctggtagccc ccgtcttaga agacgtacaa cagaaaacag ccccttacga 6420

tcgttcttct gcggcgttcg aaaaggctat ggggaagaaa actgaagccc gccaatggtg 6480

ggagtcgacg gacgacgatc aactgcgcag ccttctgatc gcgttcctgg tggacgccac 6540

taatgacacc gaggaagctg ccgcggaagc cggattaagc gaattgtaca aatcctggcc 6600

cgcggaagag cgcgaggctt taagcaatat cgacttcgaa aagggtcgcg tggcgtactc 6660

acaagaaaca ctgtccaagt taagtgaata tatgcacgaa tatcgcgtcg ggctgcatga 6720

agcacgtaaa gcagtatttg gggttgacga cacgtggcgc ccacctctgg acaaattaga 6780

agagcctacc ggacaacctg cagtagaccg tgtcctcacc atcttacgcc gcttcgtatt 6840

ggactgcgaa cgccaatggg gtcgccctcg tgccatcacc gtagagcaca ctcgcactgg 6900

tcttatgggc ccgactcaac gccaaaagat cttgaacgaa caaaagaaga atcgcgcaga 6960

caacgaacgc atccgtgacg aactgcgcga aagcggagtt gacaacccaa gtcgtgctga 7020

ggtgcgccgt cacttaatcg ttcaagaaca agaatgccaa tgtctgtatt gtggtaccat 7080

gatcacaact accaccagcg aactcgacca catcgtacca cgcgcaggcg gtggcagcag 7140

ccgccgcgaa aacttggccg ccgtatgtcg cgcctgcaac gcaaagaaga aacgcgagtt 7200

gttttacgcc tgggcgggac cggtcaaatc ccaagaaaca atcgaacgcg tgcgtcaact 7260

gaaagcattc aaagactcaa agaaagctaa gatgtttaag aaccaaatcc gccgcctgaa 7320

ccaaacagaa gcggacgaac ctatcgacga acgctctctc gcgagcacca gctacgctgc 7380

cgttgccgtc cgcgaacgct tagagcaaca tttcaacgaa ggactggccc tcgatgacaa 7440

atcacgtgtc gttttagacg tgtatgcggg cgcggtcacc cgcgaaagtc gtcgcgccgg 7500

cggcatcgat gaacgcatcc tgttgcgtgg cgaacgtgac aagaatcgct tcgacgttcg 7560

tcaccatgcc gtggacgcgg cagtaatgac gttgttgaat cgctctgtgg cattaacgct 7620

cgaacaacgt tcgcaacttc gtcgtacctt ctacgaacaa ggcttggaca agttagatcg 7680

caaccaatta aaacccgagg aagactggcg cgatttcaca gggttagccc cagccagcca 7740

agagaaattt ctggaatggc gcaaagcggc gaccatctta ggggatcttc tggccgaagc 7800

aattgaagac gatagcatcg ccgtggtatc tccgttacgc cttcgcccgc aaaatggatc 7860

tgttcactta gagaccatta gcgcggttaa gaaacagacg ctgggaagcg actggccggc 7920

agacgccgtc aaacgcatcg tcgaccctga aatctacctt gcaatgaaag acgcgctggg 7980

gaaattaaaa gaattgccgg aagactccgc tcgtagcctg gaattacctg acgggcgttt 8040

cgtggaagcg gacgacgaag tgctcttctt tcccgaaaat gcggccagca tcctgacccc 8100

gcgtggcgta gcggaaatcg gcggaagcat ccaccacgcg cgtctgtatg ggtggttaac 8160

caagaaaggt gaattaaaag ttggcatgct gcgcgtatac ggcgccgaat ttccatggct 8220

tatgcgcgaa tcgggcagcc gtaacgtgct ctctatgcct atccatcgcg gcagccaatc 8280

atttcgcgac atgcaagata ctacccgcaa ggcggtagag tccggggaag cagtagaatt 8340

cgcatggatc acgcaaaatg acgaattaga attcgaccct gacgattata tcgcgcacgg 8400

cggtaaagac gaactccgcc agttcttagg cttcatgcct gagtgtcgtt ggcgcgtcga 8460

cggcttcaag aagaattacc aaattcgcat ccgtccggcc atgttgtcgc gcgagcaact 8520

tcccagtgat atccaacgcc gcctggagag caagaccttg acgaagaatg agagccttct 8580

gttaaaggcc ttagacacgg gcttagtggt ggccatcggt ggcctgctgc cgttggagac 8640

cctgaaagtg atccgtcgta acaatctggg ttttccgcgc tggcgtggca acggtaactt 8700

gcccaccagt ttcgaggtgc gctccagcgc tctgcgcgcg cttggagtag agggg 8755

<210> 104

<211> 8599

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: plasmid(s)

<220>

<223> description: pMGX15-1 plasmid

<400> 104

ctcgagcacc accaccacca ccactgataa caaagcccga aaggaagctg agttggctgc 60

tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 120

ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc 180

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 240

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 300

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 360

gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 420

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 480

ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 540

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 600

atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 660

tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 720

atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 780

attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 840

gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 900

agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 960

aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 1020

cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 1080

cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 1140

actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 1200

cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 1260

ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 1320

ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 1380

gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 1440

gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1500

ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 1560

cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1620

caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 1680

taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 1740

cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 1800

cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 1860

gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1920

aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1980

cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2040

tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2100

acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2160

ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2220

ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2280

tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2340

tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2400

ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2460

gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2520

cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg 2580

catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg 2640

ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 2700

ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2760

ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2820

accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 2880

gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 2940

gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc 3000

cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct 3060

cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca 3120

actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt 3180

cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg 3240

gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc 3300

gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt 3360

tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg 3420

ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat 3480

ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 3540

gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 3600

gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 3660

gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 3720

tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 3780

tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3840

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 3900

tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 3960

agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 4020

tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 4080

tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 4140

gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 4200

gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 4260

tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 4320

ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 4380

gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 4440

gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 4500

gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 4560

tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 4620

cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 4680

tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 4740

tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 4800

tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 4860

cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 4920

atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 4980

ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc 5040

gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 5100

gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc 5160

ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg 5220

gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca 5280

ctataggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 5340

aaggagatat accatgaact atgtgctcgg cctggacgtc ggtattgctt cagtgggatg 5400

ggccgtgctt gagttgaatg aagaggacaa ccccattcgc attgagggtc ttggtgcgcg 5460

catcttcgat aaagcggaag tgcccaaaac cggggccagt ctcgccgcac ctcgtcgcat 5520

gagtcgtggc atccgtcgcg tgattcgtcg tcgccgcttt cgcttacaac gcgtccgctc 5580

atacctgaag aagcacaaca ttctccctgc cgagaaggtt gatcacttat acgacgttcc 5640

atctgccatc gacatctacg agctccgtaa gcgcgcactg actgagaagg tcacagccga 5700

ggagtgggcg cgcctgttaa tcttcttcgc gaaacaccgt ggtttcaagt ccaaccgcaa 5760

gaaggcttcg ggagacgccg acgagggcga gatgctcaag gcgatcgcag caaacgccga 5820

aatcctgaag aactatcgta cagtgggaga gatgctgtgc gacaacgaga agtttcgtaa 5880

acgtaagcgt aatcgcgatg gcgcatacaa ctttactgtc tctcgtgcga tgctgatgga 5940

agaaattcac acgctgttcg agattcaacg caacttgggc cagaagttcg cggacgagaa 6000

gctggaagag gagtacgtga tcctcttcgc cgctcaacgt aaattcgatg aaggacctgg 6060

agaaaattcg ccgtacgcgg ggaaccagat tgagaagatg atcggttcct gcacgttaga 6120

gggcaagaaa gagaagcgtg ctccaaaggc ctcatacgca tttatggcat ttaatttatg 6180

gcaaaagatc aatcacttga aagtcaatcg tcgtggtagt gaacgcttcc tgaccgaaga 6240

ggagcgccgc cgcattgcgg acctggcgtg gaagaaggag aagcttacct acggttcact 6300

gcgtaaggtg ctctcgttgg accccgagga tcgcttcgtt ggcctgcgtt acgacctcaa 6360

gaaaggcaag gccgagaact tagaagaacc cgtttctgag gcagaaaagc gttccttttc 6420

atgggtcaag gattatcact ccattcgtaa ggcccttgac aaggtggaga agaatcgcat 6480

tttgaagctc tctcacgagc aactggacac aattgccacc gcgtttagtg tttacaagaa 6540

cgaagagacc attgaaaagt acctggaaga gggcggcatt ggtaaggccg acggtgaggc 6600

gctgcttgag cgtttgagcg gatttagcaa gtttggtcac ctgagcctga aggcgtgtta 6660

taagctgctg ccgttcctgg agaagggcga ggtgtactca cgcgcttgtg agttggcggg 6720

gtacgacttt agtaaatcgt ccttggagaa cattgacgac attccgaacc cggtcgtaaa 6780

gcgctcgatt tctcagaccc ttaaggtgat taacgcgatc attctgcgct acggcaaccc 6840

agttgaggtt cacattgagt tagcacgtga gctcgcccgt tcttttcgcg accgcaagaa 6900

gatggagaag ggtatgcaag agaaccaagc gaagaacgag aaaattcgtc agcgcctgat 6960

tagtgagttc ggggtgctgc gcccaaaagg catggacatt gttaagttca aattatgtga 7020

ggaacaaaac ggggagtgtg cctactcgca aaagcacttt gacatggaaa agttacttaa 7080

ggacccgagt tacgccgagg ttgaccacat tatcccgtac tcacgctcat tcgacgacag 7140

ttataacaac aaggtgctgg ttctgacgaa ggagaatcgt gagaaacgta atcgtatccc 7200

gatggagtac ctcgccgacg cgcctgagcg caagaaccgt ttcattcact gggtcaaatc 7260

cacaatccgc aatagtcgca aacgtgagaa tcttctgcgt accgattaca cggcgactac 7320

cgagaatgag tggaagcgcc gtaacctgca ggacactcaa tacatttcga aatatttata 7380

taactacctg cgccatcacc tggtccttgc gaaaggttac acagagcgta agcgtcgcat 7440

cattccggtt aacggtgctg taaccgcata ctttcgcaaa cgcctgggta ttaacaagat 7500

tcgtgagaat ggcgacctgc atcacgcggt ggacgccgtt attattgcgt gtgccacaca 7560

gggaatcgtc aacaaagttt cccgctatag taaatcgcgc gaactctggg actacgaggt 7620

cgacatggag acgggtgagg tgctgcagaa gaagaacaag aataccaagg atgtctttcc 7680

ggagccgtgg cttaactttc gttacgagtt ggagcagaag gttcgtgtgc gccccttgga 7740

cattcccgag acggcagaca tcacggagat ggaagagccg ttcgttagtc atatgcctaa 7800

ccgtaagatc cacggtcctg cccacaaaga gactatccgc tctggccgcc ttaaagagga 7860

aggttacaca atcagcaaga ccgctctcat cgacttaaag cttactgagg acaaggaaga 7920

gattaagggc tattataaca aggagtcgga ccgccttttg tacgaggctt taaagaagca 7980

gctgcagcgc tacggtggca aagctaagga agcgttcaag gagccgtttc acaagcctaa 8040

agcagatggt accccgggcc caatcgttaa caaggtaaag atcatggaga agagcactat 8100

gttaatcccg gtgaacggcg ggaagggtct cgcgtcaaac ggcaacatgg ttcgcatcga 8160

cgtctttcgc gccgaggaga aaggtaagaa gaaatattac ttcatcccgg tttatgttgc 8220

tgacactgtt aaggaagagc ttccgaaccg tgcggtcttg gcccacaaac catacgaagc 8280

ctggaaaatt atgaaggaag aaaacttcat cttctccctc tacccgaacg accttatctt 8340

cgtcgacgcc ggtaaggaaa tcccgttcaa agccgcactg aagggtagca cccttgaccc 8400

ggaaaagaaa gcatcgcgtt tcctgatgta ctacaaaggg gccgacatcg ctaccggtag 8460

tattagtggc gtgaaccacg acgaaaccta caaagcacgc ggcgtaggta ttcagtcact 8520

gcgcgagatt aagaaatgtt gcattgacgt gctgggcaac atcagcttcg cttccaaaga 8580

gaagcgccaa acgttccgt 8599

<210> 105

<211> 7579

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: plasmid(s)

<220>

<223> description: pAL3 plasmid

<400> 105

ctcgagcacc accaccacca ccactgataa caaagcccga aaggaagctg agttggctgc 60

tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 120

ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc 180

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 240

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 300

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 360

gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 420

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 480

ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 540

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 600

atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 660

tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 720

atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 780

attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 840

gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 900

agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 960

aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 1020

cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 1080

cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 1140

actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 1200

cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 1260

ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 1320

ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 1380

gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 1440

gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1500

ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 1560

cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1620

caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 1680

taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 1740

cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 1800

cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 1860

gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1920

aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1980

cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2040

tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2100

acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2160

ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2220

ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2280

tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2340

tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2400

ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2460

gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2520

cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg 2580

catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg 2640

ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 2700

ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2760

ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2820

accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 2880

gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 2940

gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc 3000

cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct 3060

cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca 3120

actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt 3180

cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg 3240

gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc 3300

gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt 3360

tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg 3420

ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat 3480

ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 3540

gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 3600

gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 3660

gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 3720

tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 3780

tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3840

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 3900

tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 3960

agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 4020

tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 4080

tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 4140

gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 4200

gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 4260

tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 4320

ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 4380

gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 4440

gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 4500

gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 4560

tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 4620

cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 4680

tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 4740

tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 4800

tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 4860

cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 4920

atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 4980

ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc 5040

gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 5100

gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc 5160

ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg 5220

gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca 5280

ctataggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 5340

aaggagatat accatgtcaa ccgatatgaa gaattatcgc attggcgtgg acgtcgggga 5400

tcgcagcgta ggcttggcag cgattgagtt cgacgacgac gggctgccga ttcagaaact 5460

ggcattagtt acctttcgtc atgacggtgg cctcgaccca accaagaaca aaacgcctat 5520

gtcgcgcaaa gagacccgcg ggatcgcgcg ccgcaccatg cgtatgaatc gcgaacgcaa 5580

acgccgcctt cgcaatttgg acaacgtact ggagaactta ggctatagcg tacctgaggg 5640

acctgaaccg gaaacttacg aagcctggac gagccgcgca ctcttggcaa gcattaaatt 5700

agcatccgcc gacgaattaa acgaacacct ggttcgtgcc gttcgccaca tggcgcgcca 5760

tcgcggctgg gcaaacccct ggtggagcct ggatcaactc gaaaaggcat cacaagaacc 5820

cagcgaaacc ttcgaaatca tcttagcacg cgcacgcgaa ttattcggcg agaaagtacc 5880

agcgaacccc acccttggga tgctgggagc gctggcggca aacaacgaag tgctgctccg 5940

cccacgtgac gagaagaaac gcaaaacggg atacgtacgt ggaacgccgt tgatgttcgc 6000

acaagtgcgc caaggcgatc aattagccga attacgccgc atctgtgaag tacaaggcat 6060

cgaagatcaa tatgaagcgc tgcgtctggg tgtctttgat cacaaacatc cgtacgtccc 6120

aaaagagcgc gttggcaaag atccgttgaa cccgagcacc aaccgtacca tccgcgcaag 6180

ccttgaattc caagaatttc gtatcttaga cagtgttgcc aatctccgcg ttcgtatcgg 6240

cagtcgcgcc aaacgcgaac tcaccgaagc ggaatacgac gcagcggtag aatttctgat 6300

ggattacgcg gacaaagagc agcctagttg ggcggacgtg gcggaaaaga tcggggtgcc 6360

gggcaatcgt ctggtagccc ccgtcttaga agacgtacaa cagaaaacag ccccttacga 6420

tcgttcttct gcggcgttcg aaaaggctat ggggaagaaa actgaagccc gccaatggtg 6480

ggagtcgacg gacgacgatc aactgcgcag ccttctgatc gcgttcctgg tggacgccac 6540

taatgacacc gaggaagctg ccgcggaagc cggattaagc gaattgtaca aatcctggcc 6600

cgcggaagag cgcgaggctt taagcaatat cgacttcgaa aagggtcgcg tggcgtactc 6660

acaagaaaca ctgtccaagt taagtgaata tatgcacgaa tatcgcgtcg ggctgcatga 6720

agcacgtaaa gcagtatttg gggttgacga cacgtggcgc ccacctctgg acaaattaga 6780

agagcctacc ggacaacctg cagtagaccg tgtcctcacc atcttacgcc gcttcgtatt 6840

ggactgcgaa cgccaatggg gtcgccctcg tgccatcacc gtagagcaca ctcgcactgg 6900

tcttatgggc ccgactcaac gccaaaagat cttgaacgaa caaaagaaga atcgcgcaga 6960

caacgaacgc atccgtgacg aactgcgcga aagcggagtt gacaacccaa gtcgtgctga 7020

ggtgcgccgt cacttaatcg ttcaagaaca agaatgccaa tgtctgtatt gtggtaccat 7080

gatcacaact accaccagcg aactcgacca catcgtacca cgcgcaggcg gtggcagcag 7140

ccgccgcgaa aacttggccg ccgtatgtcg cgcctgcaac gcaaagaaga aacgcgagtt 7200

gttttacgcc tgggcgggac cggtcaaatc ccaagaaaca atcgaacgcg tgcgtcaact 7260

gaaagcattc aaagactcaa agaaagctaa gatgtttaag aaccaaatcc gccgcctgaa 7320

ccaaacagaa gcggacgaac ctatcgacga acgctctctc gcgagcacca gctacgctgc 7380

cgttgccgtc cgcgaacgct tagagcaaca tttcaacgaa ggactggccc tcgatgacaa 7440

atcacgtgtc gttttagacg tgtatgcggg cgcggtcacc cgcgaaagtc gtcgcgccgg 7500

cggcatcgat gaacgcatcc tgttgcgtgg cgaacgtgac aagaatcgct tcgacgttcg 7560

tcaccatgcc gtggacgcg 7579

<210> 106

<211> 2356

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: plasmid(s)

<220>

<223> description: PAM library U67/U40 plasmid

<220>

<221> modified_base

<222> (2277)..(2284)

<223> a, c, t, g, unknown or other

<400> 106

aatttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 60

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 120

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 180

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 240

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 300

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagccatatt 360

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 420

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 480

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 540

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 600

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 660

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 720

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 780

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 840

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 900

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 960

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 1020

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 1080

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 1140

ttgatgctcg atgagttttt ctaactgtca gaccaagttt actcatatat actttagatt 1200

gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 1260

atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 1320

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 1380

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 1440

aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 1500

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 1560

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 1620

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 1680

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 1740

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 1800

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 1860

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 1920

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 1980

atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 2040

gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 2100

gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 2160

tggcacgaca ggtttcccga ctggaaattc attaaaaatt gaattgacat taacctataa 2220

aaataggcgt cgaggccctt tcgtcttccg tgagccacca cgtcgcaagc ctcgacnnnn 2280

nnnntggaga tatcttgaac cttgcatccc cggaagagag tcaatcccgg aagagagtca 2340

attcagggtg gtgaat 2356

<210> 107

<211> 2801

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: plasmid(s)

<220>

<223> description: pMG010 plasmid

<400> 107

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 60

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 120

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 180

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 240

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 300

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 360

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 420

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 480

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 540

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 600

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 660

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 720

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 780

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 840

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 900

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 960

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1020

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1080

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1140

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1200

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1260

gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 1320

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1380

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1440

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1500

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 1560

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 1620

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 1680

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 1740

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 1800

cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 1860

gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 1920

aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 1980

actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 2040

cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 2100

aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg 2160

actctagagg atcccttgaa gactaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aagaagagca agtcccgaat tcactggccg tcgttttaca acgtcgtgac 2340

tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 2400

tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 2460

ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 2520

atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 2580

ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 2640

caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 2700

cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 2760

atggtttctt agacgtcagg tggcactttt cggggaaatg t 2801

<210> 108

<211> 3773

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 containing 5'UTR, NLS, CDS, NLS, 3' UTR, polyA tail

<220>

<223> description: MG3-6/3-4 mRNA

<400> 108

aaaagccagc tccagcaggc gctgctcact cctccccatc ctctccctct gtccctctgt 60

ccctctgacc ctgcactgtc ccagcaccat ggcccccaag aagaagcgga aagttggcgg 120

cggaggcagc tccacggata tgaagaacta caggatcggg gtcgatgtgg gagaccggtc 180

cgtgggactc gccgccatcg aatttgatga tgatggcttg cccatccaaa agctagctct 240

cgtgactttc cgacacgatg gcgggctaga tcctacaaag aataagactc ccatgagccg 300

gaaggaaaca agaggtattg caagacggac gatgaggatg aaccgggaga gaaagcggcg 360

tctgcgaaac ctggataatg tccttgaaaa tctgggatac tcagtcccag aaggcccaga 420

gccagagacc tatgaggcat ggacctcacg ggccctgctg gcttccatca agctggctag 480

cgctgatgag ctgaatgagc atttggtgcg ggctgtgcga catatggcta ggcacagagg 540

atgggcgaat ccttggtggt ccctcgacca gctggagaaa gccagccagg agccttctga 600

gacatttgag attatacttg ctagggctcg agagctgttt ggtgaaaagg ttcctgccaa 660

tcctactctg ggtatgctcg gcgccttggc agccaataat gaggttctcc tgcgaccgag 720

agatgaaaag aagaggaaga caggctatgt gcgagggact cctctgatgt ttgcgcaggt 780

ccgtcagggt gaccagcttg ctgagctgcg acggatttgc gaggtgcagg gaattgagga 840

ccagtacgag gccctcagac ttggggtgtt cgaccataag cacccctatg tgcccaagga 900

aagagtgggg aaggaccccc tcaatccatc tacaaataga acgatacggg ctagtttgga 960

gtttcaggag ttcaggatac tggattctgt agctaacctt agggtacgga ttgggtctcg 1020

tgcgaagcgg gagctgactg aggcagagta tgatgccgct gttgagttcc taatggacta 1080

tgctgataag gaacaaccat cctgggctga tgttgctgag aaaataggcg tccccgggaa 1140

ccggttggtc gctcctgtgc tcgaggatgt ccagcaaaag accgctccat atgaccgctc 1200

cagcgctgct ttcgagaaag caatgggcaa gaagaccgag gctcggcagt ggtgggaatc 1260

tacagatgat gaccagctta gatctctgct tattgcattt cttgtagatg caacaaacga 1320

tacagaagag gcggcggctg aggcaggcct ttcagagcta tataagtctt ggcctgcaga 1380

ggaacgagaa gcactaagta acattgattt cgagaaaggt agggttgcct attctcagga 1440

gaccttgtca aaactaagcg agtacatgca tgagtaccgt gtgggactcc acgaggctag 1500

gaaggccgtg ttcggcgtag atgatacctg gcgaccgccg ttggataagc tggaagaacc 1560

cactggccag ccggctgtgg atcgcgtgct aactatactg aggcggtttg tcctggattg 1620

tgagaggcag tggggtcgtc cccgagcaat aacagtggaa cataccagga ccggcttgat 1680

gggacctacc cagaggcaga aaattctgaa tgagcagaag aagaaccgag ctgataatga 1740

gcggattcgc gatgagctac gggagtctgg cgtggataat ccttctcggg ccgaagtacg 1800

gcggcatttg attgtacagg agcaggagtg tcagtgcttg tactgcggca ctatgattac 1860

gaccacaaca tcagagctgg atcatattgt gcccagggct ggggggggat cttcccgaag 1920

ggagaatctg gcagctgttt gcagagcttg taatgccaag aagaagagag aactcttcta 1980

tgcatgggct ggcccagtta agtctcagga gaccattgag agagttagac agcttaaggc 2040

ctttaaggat tctaagaagg ccaaaatgtt caagaatcag attaggcgat tgaatcagac 2100

cgaggccgat gagcccattg atgagcggtc cctggcatca acaagttatg ccgctgtggc 2160

tgtgcgagag agactagaac agcactttaa tgagggcctc gcgctggacg ataagagccg 2220

ggtggtgctt gatgtctacg ctggggccgt gacacgggag tccaggcggg caggtggtat 2280

tgacgagagg attctgctgc ggggagagcg agataagaac cgatttgatg tccgacatca 2340

cgcggtcgat gccgcagtca tgactttgct taataggagc gtcgcactca cccttgagca 2400

gagaagccag ctacgcaggg ctttctatga gctggagcta gataaacttg accgagatca 2460

gcttaagccc ggcgaagatt ggcggaactt tacgggcctc tacgaggcaa gccagaataa 2520

gttctcagag tggaagaagg ccgctacagt actcggggat ctactcgctg aagccattga 2580

ggatgatgcc attgctgtcg tgagccctct gcgattgagg ccgcagaatg gcagcgtcca 2640

cgatgacaca attaatgcgg ttaagaagct gactttgggg tccgcatggc cagccgatgc 2700

cgtgaagagg atagttgatc cagagatata cttggcaatg aaggacgtcc taggaaaact 2760

aaaggagcta cccgaggata gcgcaaggtc cttggagctc tctgatggca ggtacataga 2820

ggctgatgat gaggttctgt tcttccctaa gaaggcagct tccatactga ctccaagagg 2880

cgctgcagag atagggaatt ctatacatca tgcccgcctt tattcttggc tgaccaagaa 2940

gggagaattg aagttcggaa tgctccgagt gtatggggcg gagttcccgt ggctcatgcg 3000

ggaatccggt tccagagatg ttctccatat gcctattcat ccaggaagtc agtcattccg 3060

cggcatgcag gatggggttc gaaaggcagt agagagcggc gaagctgtgg agtttggatg 3120

gattacccag gatgatgagt tggagtttga tcctgaagac tacattgctc acggcggcga 3180

tgatgagctt aataggctac ttagagtaat gcctgagaga aggtggcggg tcgatggctt 3240

ctataatgcc ggaacgctta ggattagacc agcacttctc tctgcggagc agctgccttc 3300

agagctccag aagaaggtgg cagataagac tctgagtgac gttgagctta ttctgctgcg 3360

cgccgtacag cgtgggttgt ttgttgctat aagtagcttt ctccctctgg agagcctgaa 3420

ggttataagg cggaataact tgggcttccc aagatggcgg ggaaacggaa atttgccaac 3480

atctttcgag gtcaggagca gtgctctcag ggctctcggg gttgaaggat ctggcggaaa 3540

aagacctgcc gccacaaaga aagccggaca ggccaagaaa aagaagtgac cacaccccca 3600

ttcccccact ccagatagaa cttcagttat atctcacgtg tctggagttg gatcccttga 3660

agactaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 3773

<210> 109

<211> 1279

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG29 chimeric effector

<220>

<223> description: MG29-1_29-5 chimera

<400> 109

Met Phe Asn Asn Phe Ile Lys Lys Tyr Ser Leu Gln Lys Thr Leu Arg

1 5 10 15

Phe Glu Leu Lys Pro Val Gly Glu Thr Ala Asp Tyr Ile Glu Asp Phe

20 25 30

Lys Ser Glu Tyr Leu Lys Asp Thr Val Leu Lys Asp Glu Gln Arg Ala

35 40 45

Lys Asp Tyr Gln Glu Ile Lys Thr Leu Ile Asp Asp Tyr His Arg Glu

50 55 60

Tyr Ile Glu Glu Cys Leu Arg Glu Pro Val Asp Lys Lys Thr Gly Glu

65 70 75 80

Ile Leu Asp Phe Thr Gln Asp Leu Glu Asp Ala Phe Ser Tyr Tyr Gln

85 90 95

Lys Leu Lys Glu Asn Pro Thr Glu Asn Arg Val Gly Trp Glu Lys Glu

100 105 110

Gln Glu Ser Leu Arg Lys Lys Leu Val Thr Ser Phe Val Gly Asn Asp

115 120 125

Gly Leu Phe Lys Lys Glu Phe Ile Thr Arg Asp Leu Pro Glu Trp Leu

130 135 140

Gln Lys Lys Gly Leu Trp Gly Glu Tyr Lys Asp Thr Val Glu Asn Phe

145 150 155 160

Lys Lys Phe Thr Thr Tyr Phe Ser Gly Phe His Glu Asn Arg Lys Asn

165 170 175

Met Tyr Thr Ala Glu Ala Gln Ser Thr Ala Ile Ala Asn Arg Leu Met

180 185 190

Asn Asp Asn Leu Pro Lys Phe Phe Asn Asn Tyr Leu Ala Tyr Gln Thr

195 200 205

Ile Lys Glu Lys His Pro Asp Leu Val Phe Arg Leu Asp Asp Ala Leu

210 215 220

Leu Gln Ala Ala Gly Val Glu His Leu Asp Glu Ala Phe Gln Pro Arg

225 230 235 240

Tyr Phe Ser Arg Leu Phe Ala Gln Ser Gly Ile Thr Ala Phe Asn Glu

245 250 255

Leu Ile Gly Gly Arg Thr Thr Glu Asn Gly Glu Lys Ile Gln Gly Leu

260 265 270

Asn Glu Gln Ile Asn Leu Tyr Arg Gln Gln Asn Pro Glu Lys Ala Lys

275 280 285

Gly Phe Pro Arg Phe Met Pro Leu Phe Lys Gln Ile Leu Ser Asp Arg

290 295 300

Glu Thr His Ser Phe Leu Pro Asp Ala Phe Glu Asn Asp Lys Glu Leu

305 310 315 320

Leu Gln Ala Leu Arg Asp Tyr Val Asp Ala Ala Thr Ser Glu Glu Gly

325 330 335

Met Ile Ser Gln Leu Asn Lys Ala Met Asn Gln Phe Val Thr Ala Asp

340 345 350

Leu Lys Arg Val Tyr Ile Lys Ser Ala Ala Leu Thr Ser Leu Ser Gln

355 360 365

Glu Leu Phe His Phe Phe Gly Val Ile Ser Asp Ala Ile Ala Trp Tyr

370 375 380

Ala Glu Lys Arg Leu Ser Pro Lys Lys Ala Gln Glu Ser Phe Leu Lys

385 390 395 400

Gln Glu Val Tyr Ala Ile Glu Glu Leu Asn Gln Ala Val Val Gly Tyr

405 410 415

Ile Asp Gln Leu Glu Asp Gln Ser Glu Leu Gln Gln Leu Leu Val Asp

420 425 430

Leu Pro Asp Pro Gln Lys Pro Val Ser Ser Phe Ile Leu Thr His Trp

435 440 445

Gln Lys Ser Gln Glu Pro Leu Gln Ala Val Ile Ala Lys Val Glu Pro

450 455 460

Leu Phe Glu Leu Glu Glu Leu Ser Lys Asn Lys Arg Ala Pro Lys His

465 470 475 480

Asp Lys Asp Gln Gly Gly Glu Gly Phe Gln Gln Val Asp Ala Ile Lys

485 490 495

Asn Met Leu Asp Ala Phe Met Glu Val Ser His Ala Ile Lys Pro Leu

500 505 510

Tyr Leu Val Lys Gly Arg Lys Ala Ile Asp Met Pro Asp Val Asp Thr

515 520 525

Gly Phe Tyr Ala Asp Phe Ala Glu Ala Tyr Ser Ala Tyr Glu Gln Val

530 535 540

Thr Val Ser Leu Tyr Asn Lys Thr Arg Asn His Leu Ser Lys Lys Pro

545 550 555 560

Tyr Lys Arg Asp Lys Ile Lys Leu Asn Phe Glu Ala Pro Thr Leu Leu

565 570 575

Asn Gly Trp Asp Leu Asn Lys Glu Arg Ala Asn Arg Ser Val Leu Leu

580 585 590

Leu Lys Asn Gly Asn Tyr Tyr Leu Ala Ile Met His Pro Asn His Thr

595 600 605

Asp Ile Phe Lys Lys Tyr Met Glu Met Asp Asn Ser Asp Asn Tyr Glu

610 615 620

Lys Ile Asn Tyr Lys Leu Ile Ser Asp Ala Asn Arg Met Leu Pro Arg

625 630 635 640

Val Phe Phe Ser Lys Lys Gly Ile Lys Thr Tyr Asp Pro Pro Lys Ser

645 650 655

Ile Leu Glu Leu Tyr Lys Lys Gly Glu His Ile Lys Gly Pro Ser Phe

660 665 670

Lys Leu Glu Ser Leu His Arg Leu Ile Asp Tyr Phe Lys Ser Val Val

675 680 685

Ser Lys Tyr Lys Ala Asp Pro Gly Asp Gln Tyr Gly Trp Glu Val Phe

690 695 700

Asp Phe Lys Phe Ser Pro Thr Ser Gln Tyr Glu Asp Ile Gly Gln Phe

705 710 715 720

Tyr Lys Glu Leu Glu Lys Gln Ala Tyr Arg Val Trp Phe Thr Pro Ile

725 730 735

Ser Ser Thr Tyr Ile Glu Glu Ala Ala Lys His Gly Lys Leu Phe Leu

740 745 750

Phe Gln Ile Tyr Asn Lys Asp Phe Ser Pro Tyr Ala Lys Gly Arg Pro

755 760 765

Asn Leu His Thr Leu Tyr Trp Lys Ser Leu Phe Glu Lys Glu Asn Leu

770 775 780

Gln Asp Val Ile Thr Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg

785 790 795 800

His His Ser Ile Lys Lys Ala Asp Thr Val Ile His Lys Ala Gly Glu

805 810 815

Thr Ile Lys Asn Lys Asn Glu Asn Asn Pro Lys Gln Glu Ser Thr Phe

820 825 830

Lys His Asp Ile Ile Lys Asp Arg Arg Tyr Thr Val Asp Lys Ile Leu

835 840 845

Phe His Val Pro Ile Thr Ile Asn Phe Lys Asn Asp Lys Val Val Arg

850 855 860

Phe Asn Asp Lys Ile Asn Gly Leu Leu Ala Ala Gln Asp Asp Val His

865 870 875 880

Val Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Tyr Thr Val

885 890 895

Val Asn Gly Lys Gly Glu Val Val Glu Gln Gly Ser Leu Asn Gln Val

900 905 910

Ala Thr Asp Gln Gly Tyr Val Val Asp Tyr Gln Gln Lys Leu His Ala

915 920 925

Lys Glu Lys Glu Arg Asp Gln Ala Arg Lys Asn Trp Ser Thr Ile Glu

930 935 940

Asn Ile Lys Glu Leu Lys Ala Gly Tyr Leu Ser Gln Val Val His Lys

945 950 955 960

Leu Ala Gln Leu Ile Val Lys His Asn Ala Ile Val Cys Leu Glu Asp

965 970 975

Leu Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

980 985 990

Tyr Gln Lys Phe Glu Lys Ala Leu Ile Asp Lys Leu Asn Tyr Leu Val

995 1000 1005

Phe Lys Glu Arg Gly Ala Thr Gln Ala Gly Gly Tyr Leu Asn Ala

1010 1015 1020

Tyr Gln Leu Ala Ala Pro Phe Glu Ser Phe Glu Lys Leu Gly Lys

1025 1030 1035

Gln Thr Gly Ile Leu Tyr Tyr Val Arg Ser Asp Tyr Thr Ser Lys

1040 1045 1050

Ile Asp Pro Ala Thr Gly Phe Val Asp Phe Leu Lys Pro Lys Tyr

1055 1060 1065

Glu Ser Met Ala Lys Ser Lys Val Phe Phe Glu Ser Phe Glu Arg

1070 1075 1080

Ile Gln Trp Asn Gln Ala Lys Gly Tyr Phe Glu Phe Glu Phe Asp

1085 1090 1095

Tyr Lys Lys Met Cys Pro Ser Arg Lys Phe Gly Asp Tyr Arg Thr

1100 1105 1110

Arg Trp Val Val Cys Thr Phe Gly Asp Thr Arg Tyr Gln Asn Arg

1115 1120 1125

Arg Asn Lys Ser Ser Gly Gln Trp Glu Thr Glu Thr Ile Asp Val

1130 1135 1140

Thr Ala Gln Leu Lys Ala Leu Phe Ala Ala Tyr Gly Ile Thr Tyr

1145 1150 1155

Asn Gln Glu Asp Asn Ile Lys Asp Ala Ile Ala Ala Val Lys Tyr

1160 1165 1170

Thr Lys Phe Tyr Lys Gln Leu Tyr Trp Leu Leu Arg Leu Thr Leu

1175 1180 1185

Ser Leu Arg His Ser Val Thr Gly Thr Asp Glu Asp Phe Ile Leu

1190 1195 1200

Ser Pro Val Ala Asp Glu Asn Gly Val Phe Phe Asp Ser Arg Lys

1205 1210 1215

Ala Thr Asp Lys Gln Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr

1220 1225 1230

His Ile Ala Leu Lys Gly Leu Trp Asn Leu Gln Gln Ile Arg Gln

1235 1240 1245

His Asp Trp Asn Val Glu Lys Pro Lys Lys Leu Asn Leu Ala Met

1250 1255 1260

Lys Asn Glu Glu Trp Phe Gly Phe Ala Gln Lys Lys Lys Phe Arg

1265 1270 1275

Ala

<210> 110

<211> 1291

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG29 chimeric effector

<220>

<223> description: MG29-1_57-1 chimera

<400> 110

Met Phe Asn Asn Phe Ile Lys Lys Tyr Ser Leu Gln Lys Thr Leu Arg

1 5 10 15

Phe Glu Leu Lys Pro Val Gly Glu Thr Ala Asp Tyr Ile Glu Asp Phe

20 25 30

Lys Ser Glu Tyr Leu Lys Asp Thr Val Leu Lys Asp Glu Gln Arg Ala

35 40 45

Lys Asp Tyr Gln Glu Ile Lys Thr Leu Ile Asp Asp Tyr His Arg Glu

50 55 60

Tyr Ile Glu Glu Cys Leu Arg Glu Pro Val Asp Lys Lys Thr Gly Glu

65 70 75 80

Ile Leu Asp Phe Thr Gln Asp Leu Glu Asp Ala Phe Ser Tyr Tyr Gln

85 90 95

Lys Leu Lys Glu Asn Pro Thr Glu Asn Arg Val Gly Trp Glu Lys Glu

100 105 110

Gln Glu Ser Leu Arg Lys Lys Leu Val Thr Ser Phe Val Gly Asn Asp

115 120 125

Gly Leu Phe Lys Lys Glu Phe Ile Thr Arg Asp Leu Pro Glu Trp Leu

130 135 140

Gln Lys Lys Gly Leu Trp Gly Glu Tyr Lys Asp Thr Val Glu Asn Phe

145 150 155 160

Lys Lys Phe Thr Thr Tyr Phe Ser Gly Phe His Glu Asn Arg Lys Asn

165 170 175

Met Tyr Thr Ala Glu Ala Gln Ser Thr Ala Ile Ala Asn Arg Leu Met

180 185 190

Asn Asp Asn Leu Pro Lys Phe Phe Asn Asn Tyr Leu Ala Tyr Gln Thr

195 200 205

Ile Lys Glu Lys His Pro Asp Leu Val Phe Arg Leu Asp Asp Ala Leu

210 215 220

Leu Gln Ala Ala Gly Val Glu His Leu Asp Glu Ala Phe Gln Pro Arg

225 230 235 240

Tyr Phe Ser Arg Leu Phe Ala Gln Ser Gly Ile Thr Ala Phe Asn Glu

245 250 255

Leu Ile Gly Gly Arg Thr Thr Glu Asn Gly Glu Lys Ile Gln Gly Leu

260 265 270

Asn Glu Gln Ile Asn Leu Tyr Arg Gln Gln Asn Pro Glu Lys Ala Lys

275 280 285

Gly Phe Pro Arg Phe Met Pro Leu Phe Lys Gln Ile Leu Ser Asp Arg

290 295 300

Glu Thr His Ser Phe Leu Pro Asp Ala Phe Glu Asn Asp Lys Glu Leu

305 310 315 320

Leu Gln Ala Leu Arg Asp Tyr Val Asp Ala Ala Thr Ser Glu Glu Gly

325 330 335

Met Ile Ser Gln Leu Asn Lys Ala Met Asn Gln Phe Val Thr Ala Asp

340 345 350

Leu Lys Arg Val Tyr Ile Lys Ser Ala Ala Leu Thr Ser Leu Ser Gln

355 360 365

Glu Leu Phe His Phe Phe Gly Val Ile Ser Asp Ala Ile Ala Trp Tyr

370 375 380

Ala Glu Lys Arg Leu Ser Pro Lys Lys Ala Gln Glu Ser Phe Leu Lys

385 390 395 400

Gln Glu Val Tyr Ala Ile Glu Glu Leu Asn Gln Ala Val Val Gly Tyr

405 410 415

Ile Asp Gln Leu Glu Asp Gln Ser Glu Leu Gln Gln Leu Leu Val Asp

420 425 430

Leu Pro Asp Pro Gln Lys Pro Val Ser Ser Phe Ile Leu Thr His Trp

435 440 445

Gln Lys Ser Gln Glu Pro Leu Gln Ala Val Ile Ala Lys Val Glu Pro

450 455 460

Leu Phe Glu Leu Glu Glu Leu Ser Lys Asn Lys Arg Ala Pro Lys His

465 470 475 480

Asp Lys Asp Gln Gly Gly Glu Gly Phe Gln Gln Val Asp Ala Ile Lys

485 490 495

Asn Met Leu Asp Ala Phe Met Glu Val Ser His Ala Ile Lys Pro Leu

500 505 510

Tyr Leu Val Lys Gly Arg Lys Ala Ile Asp Met Pro Asp Val Asp Thr

515 520 525

Gly Phe Tyr Ala Asp Phe Ala Glu Ala Tyr Ser Ala Tyr Glu Gln Val

530 535 540

Thr Val Ser Leu Tyr Asn Lys Thr Arg Asn His Leu Ser Lys Lys Pro

545 550 555 560

Gln Asp Asp Val Lys Glu Asn Lys Leu Lys Leu Asn Phe Gly Asn Ser

565 570 575

Ser Leu Leu Gly Gly Trp Ser Asp Gly Gln Glu Lys Thr Lys Ala Ala

580 585 590

Thr Leu Leu Arg Cys Glu Asn Asp Leu Tyr Leu Cys Ile Leu Lys Lys

595 600 605

Arg Asp Val Phe Asp Thr Ser Lys Asp Asn Pro Ile Tyr Ser Ser Pro

610 615 620

Ser Asn Ala Ser Arg Leu Ile Ile Arg Asn Leu Lys Phe Gln Thr Leu

625 630 635 640

Ala Gly Lys Gly Phe Leu Ser Glu Tyr Gly Ile Ser Tyr Gly Glu Met

645 650 655

Ser Lys Asp Asp Pro Lys Lys Ala Ile Gln Cys Leu Gln Glu Ile Ile

660 665 670

Lys Glu Arg Tyr Val Lys Lys Tyr Pro Leu Leu Glu Lys Phe Val Thr

675 680 685

Ile Lys Tyr Ala Asp Lys Ser Glu Phe Asp Ala Glu Ile Asn Glu Thr

690 695 700

Leu Lys Glu Cys Tyr Val Cys Glu Phe Val Pro Ile Asn Trp Asn Leu

705 710 715 720

Ile Val Glu Lys Gln Asn Asn Asp Glu Leu Phe Leu Phe Lys Ile Leu

725 730 735

Cys Lys Asp Tyr Lys Pro Asn Ser Ile Gly Lys Arg Asp Leu Gln Thr

740 745 750

Ile Tyr Trp Glu Asp Val Leu Ser Asp Ala Ser Asn His Gln Leu Cys

755 760 765

Ala Gly Ala Glu Ile Phe Met Arg Lys Pro Ile Ala Asn Glu Ser Pro

770 775 780

Leu Thr His Arg Ile Gly Ser Lys Leu Val Asn Lys Arg Asp Lys Asp

785 790 795 800

Gly Asn Thr Ile Pro Asp Lys Phe Tyr Arg Glu Ile Tyr Leu Tyr Ala

805 810 815

Asn Gly Lys Thr Lys Val Ile Ser Lys Glu Ala Lys Lys Tyr Ile Asp

820 825 830

Glu Glu Lys Ala Thr Ile Lys Asp Val Asn His Glu Ile Ile Lys Asp

835 840 845

Lys Arg Phe Tyr Gly Asp Ser Gln Glu Pro Gln Tyr Met Phe His Cys

850 855 860

Pro Ile Lys Leu Asn Tyr Lys Thr Lys Val Val Arg Phe Asn Asp Lys

865 870 875 880

Ile Asn Gly Leu Leu Ala Ala Gln Asp Asp Val His Val Ile Gly Ile

885 890 895

Asp Arg Gly Glu Arg His Leu Leu Tyr Tyr Thr Val Val Asn Gly Lys

900 905 910

Gly Glu Val Val Glu Gln Gly Ser Leu Asn Gln Val Ala Thr Asp Gln

915 920 925

Gly Tyr Val Val Asp Tyr Gln Gln Lys Leu His Ala Lys Glu Lys Glu

930 935 940

Arg Asp Gln Ala Arg Lys Asn Trp Ser Thr Ile Glu Asn Ile Lys Glu

945 950 955 960

Leu Lys Ala Gly Tyr Leu Ser Gln Val Val His Lys Leu Ala Gln Leu

965 970 975

Ile Val Lys His Asn Ala Ile Val Cys Leu Glu Asp Leu Asn Phe Gly

980 985 990

Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Phe

995 1000 1005

Glu Lys Ala Leu Ile Asp Lys Leu Asn Tyr Leu Val Phe Lys Glu

1010 1015 1020

Arg Gly Ala Thr Gln Ala Gly Gly Tyr Leu Asn Ala Tyr Gln Leu

1025 1030 1035

Ala Ala Pro Phe Glu Ser Phe Glu Lys Leu Gly Lys Gln Thr Gly

1040 1045 1050

Ile Leu Tyr Tyr Val Arg Ser Asp Tyr Thr Ser Lys Ile Asp Pro

1055 1060 1065

Ala Thr Gly Phe Val Asp Phe Leu Lys Pro Lys Tyr Glu Ser Met

1070 1075 1080

Ala Lys Ser Lys Val Phe Phe Glu Ser Phe Glu Arg Ile Gln Trp

1085 1090 1095

Asn Gln Ala Lys Gly Tyr Phe Glu Phe Glu Phe Asp Tyr Lys Lys

1100 1105 1110

Met Cys Pro Ser Arg Lys Phe Gly Asp Tyr Arg Thr Arg Trp Val

1115 1120 1125

Val Cys Thr Phe Gly Asp Thr Arg Tyr Gln Asn Arg Arg Asn Lys

1130 1135 1140

Ser Ser Gly Gln Trp Glu Thr Glu Thr Ile Asp Val Thr Ala Gln

1145 1150 1155

Leu Lys Ala Leu Phe Ala Ala Tyr Gly Ile Thr Tyr Asn Gln Glu

1160 1165 1170

Asp Asn Ile Lys Asp Ala Ile Ala Ala Val Lys Tyr Thr Lys Phe

1175 1180 1185

Tyr Lys Gln Leu Tyr Trp Leu Leu Arg Leu Thr Leu Ser Leu Arg

1190 1195 1200

His Ser Val Thr Gly Thr Asp Glu Asp Phe Ile Leu Ser Pro Val

1205 1210 1215

Ala Asp Glu Asn Gly Val Phe Phe Asp Ser Arg Lys Ala Thr Asp

1220 1225 1230

Lys Gln Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Ala

1235 1240 1245

Leu Lys Gly Leu Trp Asn Leu Gln Gln Ile Arg Gln His Asp Trp

1250 1255 1260

Asn Val Glu Lys Pro Lys Lys Leu Asn Leu Ala Met Lys Asn Glu

1265 1270 1275

Glu Trp Phe Gly Phe Ala Gln Lys Lys Lys Phe Arg Ala

1280 1285 1290

<210> 111

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG29 sgRNA

<220>

Description of: MG29-1 sgRNA

<220>

<221> modified_base

<222> (20)..(39)

<223> a, c, t, g, unknown or other

<400> 111

aatttctact gttgtagatn nnnnnnnnnn nnnnnnnnn 39

<210> 112

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG29 sgRNA

<220>

Description of: MG29-5 sgRNA

<220>

<221> modified_base

<222> (20)..(39)

<223> a, c, t, g, unknown or other

<400> 112

aatttctaca gttgtagatn nnnnnnnnnn nnnnnnnnn 39

<210> 113

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG57 sgRNA

<220>

<223> description: MG57-1 sgRNA

<220>

<221> modified_base

<222> (20)..(39)

<223> a, c, t, g, unknown or other

<400> 113

aatttctact atcgtagatn nnnnnnnnnn nnnnnnnnn 39

<210> 114

<211> 4

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG29 PAM

<220>

<223> description: MG29-1 PAM

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, t, g, unknown or other

<400> 114

tttn 4

<210> 115

<211> 4

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG29 PAM

<220>

<223> description: MG29-5 PAM

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, t, g, unknown or other

<400> 115

yyyn 4

<210> 116

<211> 2

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG57 PAM

<220>

<223> description: MG57-1 PAM

<220>

<221> modified_base

<222> (2)..(2)

<223> a, c, t, g, unknown or other

<400> 116

yn 2

<210> 117

<211> 4

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: MG29 chimeric PAM

<220>

<223> description: MG29-1_29-5 PAM

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, t, g, unknown or other

<400> 117

tyyn 4

<210> 118

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: primer(s)

<220>

<223> description: LA179 primer for PCR in PAM enrichment assay

<220>

<221> modified_base

<222> (14)..(18)

<223> a, c, t, g, unknown or other

<400> 118

gctcttccga tctnnnnngc agctggcacg acagg 35

<210> 119

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC A1

<400> 119

gccguguacc agcugagaga cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 120

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC B1

<400> 120

auucaccgau uuugauucuc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 121

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC C1

<400> 121

gauucugaug uguauaucac agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 122

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC D1

<400> 122

aacagugcug uggccuggag caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 123

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC E1

<400> 123

ggcuggggaa gaaggugucu ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 124

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC F1

<400> 124

guuuugucug ugauauacac auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 125

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC G1

<400> 125

uuacuuugug acacauuugu uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 126

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC H1

<400> 126

uugugacaca uuuguuugag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 127

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC A2

<400> 127

ugugacacau uuguuugaga auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 128

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC B2

<400> 128

auuuguuuga gaaucaaaau cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 129

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC C2

<400> 129

uuccugugau gucaagcugg ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 130

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC D2

<400> 130

uccugugaug ucaagcuggu cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 131

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC E2

<400> 131

gucaagcugg ucgagaaaag cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 132

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC F2

<400> 132

agcuugacau cacaggaacu uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 133

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC G2

<400> 133

gacaucacag gaacuuucua aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 134

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC H2

<400> 134

uuacagauac gaaccuaaac uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 135

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC A3

<400> 135

aaaaccuguc agugauuggg uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 136

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC B3

<400> 136

gauuggguuc cgaauccucc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 137

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC C3

<400> 137

ggaacccaau cacugacagg uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 138

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRAC-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRAC D3

<400> 138

uugaaaguuu agguucguau cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 139

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC A1

<400> 139

gccgtgtacc agctgagaga ct 22

<210> 140

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC B1

<400> 140

attcaccgat tttgattctc aa 22

<210> 141

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC C1

<400> 141

gattctgatg tgtatatcac ag 22

<210> 142

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC D1

<400> 142

aacagtgctg tggcctggag ca 22

<210> 143

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC E1

<400> 143

ggctggggaa gaaggtgtct tc 22

<210> 144

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC F1

<400> 144

gttttgtctg tgatatacac at 22

<210> 145

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC G1

<400> 145

ttactttgtg acacatttgt tt 22

<210> 146

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC H1

<400> 146

ttgtgacaca tttgtttgag aa 22

<210> 147

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC A2

<400> 147

tgtgacacat ttgtttgaga at 22

<210> 148

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC B2

<400> 148

atttgtttga gaatcaaaat cg 22

<210> 149

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC C2

<400> 149

ttcctgtgat gtcaagctgg tc 22

<210> 150

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC D2

<400> 150

tcctgtgatg tcaagctggt cg 22

<210> 151

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC E2

<400> 151

gtcaagctgg tcgagaaaag ct 22

<210> 152

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC F2

<400> 152

agcttgacat cacaggaact tt 22

<210> 153

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC G2

<400> 153

gacatcacag gaactttcta aa 22

<210> 154

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC H2

<400> 154

ttacagatac gaacctaaac tt 22

<210> 155

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC A3

<400> 155

aaaacctgtc agtgattggg tt 22

<210> 156

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC B3

<400> 156

gattgggttc cgaatcctcc tc 22

<210> 157

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC C3

<400> 157

ggaacccaat cactgacagg tt 22

<210> 158

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRAC target site

<220>

<223> description: MG3-6/3-4 TRAC D3

<400> 158

ttgaaagttt aggttcgtat ct 22

<210> 159

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M A1

<400> 159

ucacgcugga uagccuccag gcguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 160

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M B1

<400> 160

gguuuacuca cgucauccag caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 161

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M C1

<400> 161

acucacguca uccagcagag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 162

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M D1

<400> 162

ucauccagca gagaauggaa agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 163

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M E1

<400> 163

agagaaugga aagucaaauu ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 164

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M F1

<400> 164

cgacauugaa guugacuuac ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 165

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M G1

<400> 165

uugacuuacu gaagaaugga gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 166

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M H1

<400> 166

uuacugaaga auggagagag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 167

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M A2

<400> 167

uacugaagaa uggagagaga auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 168

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 B2M B2

<400> 168

acugaagaau ggagagagaa uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 169

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M C2

<400> 169

ucuuucuauc ucuuguacua caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 170

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M D2

<400> 170

uacuacacug aauucacccc caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 171

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M E2

<400> 171

acuacacuga auucaccccc acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 172

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M F2

<400> 172

cuacacugaa uucaccccca cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 173

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M G2

<400> 173

auacucaucu uuuucagugg ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 174

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M H2

<400> 174

gaauucagug uaguacaaga gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 175

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M A3

<400> 175

gagauagaaa gaccaguccu ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 176

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M B3

<400> 176

caguccuugc ugaaagacaa guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 177

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M C3

<400> 177

agucaacuuc aaugucggau ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 178

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M D3

<400> 178

aaacccagac acauagcaau ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 179

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M E3

<400> 179

aacccagaca cauagcaauu caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 180

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M F3

<400> 180

cugcuggaug acgugaguaa acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 181

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M G3

<400> 181

accugaaucu uuggaguacc ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 182

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M H3

<400> 182

ugcugcuuac augucucgau cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 183

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M A4

<400> 183

gcugcuuaca ugucucgauc uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 184

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: B2M-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG 3-6/3-4B 2M B4

<400> 184

cugcuuacau gucucgaucu auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 185

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M A1

<400> 185

tcacgctgga tagcctccag gc 22

<210> 186

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M B1

<400> 186

ggtttactca cgtcatccag ca 22

<210> 187

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M C1

<400> 187

actcacgtca tccagcagag aa 22

<210> 188

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M D1

<400> 188

tcatccagca gagaatggaa ag 22

<210> 189

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M E1

<400> 189

agagaatgga aagtcaaatt tc 22

<210> 190

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M F1

<400> 190

cgacattgaa gttgacttac tg 22

<210> 191

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M G1

<400> 191

ttgacttact gaagaatgga ga 22

<210> 192

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M H1

<400> 192

ttactgaaga atggagagag aa 22

<210> 193

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M A2

<400> 193

tactgaagaa tggagagaga at 22

<210> 194

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG3-6/3-4 B2M B2

<400> 194

actgaagaat ggagagagaa tt 22

<210> 195

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M C2

<400> 195

tctttctatc tcttgtacta ca 22

<210> 196

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M D2

<400> 196

tactacactg aattcacccc ca 22

<210> 197

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M E2

<400> 197

actacactga attcaccccc ac 22

<210> 198

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M F2

<400> 198

ctacactgaa ttcaccccca ct 22

<210> 199

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M G2

<400> 199

atactcatct ttttcagtgg gg 22

<210> 200

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M H2

<400> 200

gaattcagtg tagtacaaga ga 22

<210> 201

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M A3

<400> 201

gagatagaaa gaccagtcct tg 22

<210> 202

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M B3

<400> 202

cagtccttgc tgaaagacaa gt 22

<210> 203

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M C3

<400> 203

agtcaacttc aatgtcggat gg 22

<210> 204

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M D3

<400> 204

aaacccagac acatagcaat tc 22

<210> 205

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M E3

<400> 205

aacccagaca catagcaatt ca 22

<210> 206

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M F3

<400> 206

ctgctggatg acgtgagtaa ac 22

<210> 207

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M G3

<400> 207

acctgaatct ttggagtacc tg 22

<210> 208

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M H3

<400> 208

tgctgcttac atgtctcgat ct 22

<210> 209

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M A4

<400> 209

gctgcttaca tgtctcgatc ta 22

<210> 210

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of B2M target site

<220>

<223> description: MG 3-6/3-4B 2M B4

<400> 210

ctgcttacat gtctcgatct at 22

<210> 211

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A1

<400> 211

cagaagcaga gaucucccac acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 212

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 B1

<400> 212

ccacguggag cugagcuggu ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 213

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 C1

<400> 213

aguccaguuc uacgggcucu cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 214

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 D1

<400> 214

gauuagguga gaccagcuac caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 215

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 E1

<400> 215

auuaggugag accagcuacc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 216

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 F1

<400> 216

uuaggugaga ccagcuacca ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 217

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 G1

<400> 217

ugagaccagc uaccagggaa aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 218

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 H1

<400> 218

cagguagcag acaagacuag auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 219

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A2

<400> 219

agguagcaga caagacuaga ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 220

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 B2

<400> 220

agcagacaag acuagaucca aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 221

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 C2

<400> 221

ggaaccagcg cacaccauga agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 222

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 D2

<400> 222

guggcugaca ucugcauggc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 223

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 E2

<400> 223

ggccugggag ucugugccaa cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 224

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 F2

<400> 224

cugacuuuac uuuuaauugc cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 225

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 G2

<400> 225

ugacuuuacu uuuaauugcc uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 226

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 H2

<400> 226

gacuuuacuu uuaauugccu auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 227

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A3

<400> 227

gggaaggaga agcuggaguc acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 228

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 B3

<400> 228

ggaaggagaa gcuggaguca ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 229

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 C3

<400> 229

aacuccuggc ucuuaauaac ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 230

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 D3

<400> 230

aacuuucucu ucugcagguc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 231

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 E3

<400> 231

acuccacuuc cagggcugcc uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 232

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 F3

<400> 232

cuccacuucc agggcugccu ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 233

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 G3

<400> 233

uccuuucucu ugaccugcag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 234

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 H3

<400> 234

agccaggagu ugugaggauu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 235

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A4

<400> 235

aguaguaggg cccauugacc acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 236

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 B4

<400> 236

ugcaaguuau cuucugaggc acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 237

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 C4

<400> 237

aguuaucuuc ugaggcaccu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 238

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 D4

<400> 238

guuaucuucu gaggcaccug aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 239

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 E4

<400> 239

ucaagaacca ugagagaggg agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 240

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 F4

<400> 240

caagaaccau gagagaggga gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 241

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 G4

<400> 241

uuacccgagg uaaagccaca guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 242

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 H4

<400> 242

ccgagguaaa gccacagucu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 243

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A5

<400> 243

cagucugaaa gaaagcaggg agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 244

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 B5

<400> 244

agucugaaag aaagcaggga gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 245

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 C5

<400> 245

gucugaaaga aagcagggag agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 246

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 D5

<400> 246

gaaagaaagc agggagagga aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 247

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 E5

<400> 247

gagaccuuau uuucauaggc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 248

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 F5

<400> 248

gaugagaguu acacaggcca caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 249

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 G5

<400> 249

agcugcuugg cucuguuggg cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 250

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 H5

<400> 250

uguugggcug agaaucuggg agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 251

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 1-targeted MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC1 A6

<400> 251

ggaacaccuu guucaggucc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 252

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A1

<400> 252

cagaagcaga gatctcccac ac 22

<210> 253

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 B1

<400> 253

ccacgtggag ctgagctggt gg 22

<210> 254

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 C1

<400> 254

agtccagttc tacgggctct cg 22

<210> 255

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 D1

<400> 255

gattaggtga gaccagctac ca 22

<210> 256

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 E1

<400> 256

attaggtgag accagctacc ag 22

<210> 257

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 F1

<400> 257

ttaggtgaga ccagctacca gg 22

<210> 258

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 G1

<400> 258

tgagaccagc taccagggaa aa 22

<210> 259

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 H1

<400> 259

caggtagcag acaagactag at 22

<210> 260

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A2

<400> 260

aggtagcaga caagactaga tc 22

<210> 261

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 B2

<400> 261

agcagacaag actagatcca aa 22

<210> 262

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 C2

<400> 262

ggaaccagcg cacaccatga ag 22

<210> 263

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 D2

<400> 263

gtggctgaca tctgcatggc ag 22

<210> 264

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 E2

<400> 264

ggcctgggag tctgtgccaa ct 22

<210> 265

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 F2

<400> 265

ctgactttac ttttaattgc ct 22

<210> 266

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 G2

<400> 266

tgactttact tttaattgcc ta 22

<210> 267

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 H2

<400> 267

gactttactt ttaattgcct at 22

<210> 268

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A3

<400> 268

gggaaggaga agctggagtc ac 22

<210> 269

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 B3

<400> 269

ggaaggagaa gctggagtca cc 22

<210> 270

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 C3

<400> 270

aactcctggc tcttaataac cc 22

<210> 271

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 D3

<400> 271

aactttctct tctgcaggtc aa 22

<210> 272

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 E3

<400> 272

actccacttc cagggctgcc tt 22

<210> 273

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 F3

<400> 273

ctccacttcc agggctgcct tc 22

<210> 274

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 G3

<400> 274

tcctttctct tgacctgcag aa 22

<210> 275

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 H3

<400> 275

agccaggagt tgtgaggatt ga 22

<210> 276

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A4

<400> 276

agtagtaggg cccattgacc ac 22

<210> 277

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 B4

<400> 277

tgcaagttat cttctgaggc ac 22

<210> 278

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 C4

<400> 278

agttatcttc tgaggcacct ga 22

<210> 279

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 D4

<400> 279

gttatcttct gaggcacctg aa 22

<210> 280

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 E4

<400> 280

tcaagaacca tgagagaggg ag 22

<210> 281

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 F4

<400> 281

caagaaccat gagagaggga ga 22

<210> 282

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 G4

<400> 282

ttacccgagg taaagccaca gt 22

<210> 283

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 H4

<400> 283

ccgaggtaaa gccacagtct ga 22

<210> 284

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A5

<400> 284

cagtctgaaa gaaagcaggg ag 22

<210> 285

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 B5

<400> 285

agtctgaaag aaagcaggga ga 22

<210> 286

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 C5

<400> 286

gtctgaaaga aagcagggag ag 22

<210> 287

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 D5

<400> 287

gaaagaaagc agggagagga aa 22

<210> 288

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 E5

<400> 288

gagaccttat tttcataggc aa 22

<210> 289

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 F5

<400> 289

gatgagagtt acacaggcca ca 22

<210> 290

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 G5

<400> 290

agctgcttgg ctctgttggg ct 22

<210> 291

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 H5

<400> 291

tgttgggctg agaatctggg ag 22

<210> 292

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC1 target site

<220>

<223> description: MG3-6/3-4 TRBC1 A6

<400> 292

ggaacacctt gttcaggtcc tc 22

<210> 293

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A1

<400> 293

accucuuccc uuuccagagg acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 294

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B1

<400> 294

ccucuucccu uuccagagga ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 295

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C1

<400> 295

cucuucccuu uccagaggac cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 296

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D1

<400> 296

cagaagcaga gaucucccac acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 297

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E1

<400> 297

ccacguggag cugagcuggu ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 298

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 F1

<400> 298

aguccaguuc uacgggcucu cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 299

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 G1

<400> 299

gauuagguga gaccagcuac caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 300

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 H1

<400> 300

auuaggugag accagcuacc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 301

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A2

<400> 301

uuaggugaga ccagcuacca ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 302

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B2

<400> 302

ugagaccagc uaccagggaa aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 303

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C2

<400> 303

uagcggacaa gacuagaucc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 304

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D2

<400> 304

cccccaccaa gaagcauaga ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 305

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E2

<400> 305

ucugcucucg aaccagggca ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 306

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 F2

<400> 306

ggaacaucac acaugggcau aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 307

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 G2

<400> 307

ccuaauauau ccuaucaccu caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 308

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 H2

<400> 308

accauaauga agccagacug ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 309

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A3

<400> 309

ccauaaugaa gccagacugg ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 310

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B3

<400> 310

cauaaugaag ccagacuggg gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 311

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C3

<400> 311

gccagacugg ggagaaaaug caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 312

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D3

<400> 312

ggagaaaaug cagggaauau caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 313

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E3

<400> 313

ggagacaacc agcgagcccu acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 314

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 F3

<400> 314

uacuccugcu gugccauagc ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 315

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 G3

<400> 315

cugugccaua gccccugaaa ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 316

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 H3

<400> 316

ugugccauag ccccugaaac ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 317

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A4

<400> 317

gugccauagc cccugaaacc cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 318

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B4

<400> 318

uguucucucu uccacagguc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 319

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C4

<400> 319

gaaaggauuc cagaggcuag cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 320

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D4

<400> 320

ggaugguuuu ggagcuagcc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 321

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E4

<400> 321

cccugguucg agagcagaga cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 322

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 F4

<400> 322

agcagagacg gcgaaagaua gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 323

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 G4

<400> 323

gcagagacgg cgaaagauag agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 324

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 H4

<400> 324

cagagacggc gaaagauaga gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 325

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A5

<400> 325

uuaccggagg ugaagccaca guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 326

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B5

<400> 326

cggaggugaa gccacagucu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 327

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C5

<400> 327

ggaggugaag ccacagucug aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 328

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D5

<400> 328

acagucugaa agaaaacagg ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 329

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E5

<400> 329

cagucugaaa gaaaacaggg gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 330

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 F5

<400> 330

agucugaaag aaaacagggg aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 331

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 G5

<400> 331

gucugaaaga aaacagggga agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 332

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 H5

<400> 332

acaggggaag aaaaauggau gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 333

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 A6

<400> 333

gcgaaguggu cacuaugauc uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 334

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 B6

<400> 334

uuaggaaacc aggaccccag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 335

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 C6

<400> 335

uauggcuggu ccucagggag acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 336

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 D6

<400> 336

cuaagguguc aggaucugaa ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 337

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: TRBC 2-targeting MG3-6/3-4 sgRNA

<220>

<223> description: MG3-6/3-4 TRBC2 E6

<400> 337

ggaacacguu uuucaggucc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 338

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A1

<400> 338

acctcttccc tttccagagg ac 22

<210> 339

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B1

<400> 339

cctcttccct ttccagagga cc 22

<210> 340

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C1

<400> 340

ctcttccctt tccagaggac ct 22

<210> 341

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D1

<400> 341

cagaagcaga gatctcccac ac 22

<210> 342

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E1

<400> 342

ccacgtggag ctgagctggt gg 22

<210> 343

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 F1

<400> 343

agtccagttc tacgggctct cg 22

<210> 344

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 G1

<400> 344

gattaggtga gaccagctac ca 22

<210> 345

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 H1

<400> 345

attaggtgag accagctacc ag 22

<210> 346

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A2

<400> 346

ttaggtgaga ccagctacca gg 22

<210> 347

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B2

<400> 347

tgagaccagc taccagggaa aa 22

<210> 348

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C2

<400> 348

tagcggacaa gactagatcc ag 22

<210> 349

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D2

<400> 349

cccccaccaa gaagcataga gg 22

<210> 350

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E2

<400> 350

tctgctctcg aaccagggca tg 22

<210> 351

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 F2

<400> 351

ggaacatcac acatgggcat aa 22

<210> 352

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 G2

<400> 352

cctaatatat cctatcacct ca 22

<210> 353

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 H2

<400> 353

accataatga agccagactg gg 22

<210> 354

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A3

<400> 354

ccataatgaa gccagactgg gg 22

<210> 355

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B3

<400> 355

cataatgaag ccagactggg ga 22

<210> 356

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C3

<400> 356

gccagactgg ggagaaaatg ca 22

<210> 357

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D3

<400> 357

ggagaaaatg cagggaatat ca 22

<210> 358

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E3

<400> 358

ggagacaacc agcgagccct ac 22

<210> 359

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 F3

<400> 359

tactcctgct gtgccatagc cc 22

<210> 360

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 G3

<400> 360

ctgtgccata gcccctgaaa cc 22

<210> 361

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 H3

<400> 361

tgtgccatag cccctgaaac cc 22

<210> 362

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A4

<400> 362

gtgccatagc ccctgaaacc ct 22

<210> 363

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B4

<400> 363

tgttctctct tccacaggtc aa 22

<210> 364

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C4

<400> 364

gaaaggattc cagaggctag ct 22

<210> 365

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D4

<400> 365

ggatggtttt ggagctagcc tc 22

<210> 366

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E4

<400> 366

ccctggttcg agagcagaga cg 22

<210> 367

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 F4

<400> 367

agcagagacg gcgaaagata ga 22

<210> 368

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 G4

<400> 368

gcagagacgg cgaaagatag ag 22

<210> 369

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 H4

<400> 369

cagagacggc gaaagataga ga 22

<210> 370

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A5

<400> 370

ttaccggagg tgaagccaca gt 22

<210> 371

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B5

<400> 371

cggaggtgaa gccacagtct ga 22

<210> 372

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C5

<400> 372

ggaggtgaag ccacagtctg aa 22

<210> 373

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D5

<400> 373

acagtctgaa agaaaacagg gg 22

<210> 374

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E5

<400> 374

cagtctgaaa gaaaacaggg ga 22

<210> 375

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 F5

<400> 375

agtctgaaag aaaacagggg aa 22

<210> 376

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 G5

<400> 376

gtctgaaaga aaacagggga ag 22

<210> 377

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 H5

<400> 377

acaggggaag aaaaatggat ga 22

<210> 378

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 A6

<400> 378

gcgaagtggt cactatgatc tt 22

<210> 379

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 B6

<400> 379

ttaggaaacc aggaccccag aa 22

<210> 380

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 C6

<400> 380

tatggctggt cctcagggag ac 22

<210> 381

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 D6

<400> 381

ctaaggtgtc aggatctgaa gg 22

<210> 382

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of TRBC2 target site

<220>

<223> description: MG3-6/3-4 TRBC2 E6

<400> 382

ggaacacgtt tttcaggtcc tc 22

<210> 383

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A1

<400> 383

uuguuccucu aguuauuucc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 384

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B1

<400> 384

auuugauucu cuaucuccag agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 385

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C1

<400> 385

uuugauucuc uaucuccaga gcguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 386

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D1

<400> 386

aagauuugcu auguuagacg auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 387

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E1

<400> 387

agauuugcua uguuagacga ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 388

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F1

<400> 388

gauuugcuau guuagacgau guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 389

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G1

<400> 389

acuuugucca uaagacgaag ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 390

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H1

<400> 390

agggccaaau uaaugacaua uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 391

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A2

<400> 391

gggccaaauu aaugacauau uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 392

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B2

<400> 392

uaugaucuau cgcugcaaac caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 393

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C2

<400> 393

augaucuauc gcugcaaacc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 394

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D2

<400> 394

caaaccagug aaaucaaaga agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 395

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E2

<400> 395

aaaccaguga aaucaaagaa gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 396

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F2

<400> 396

acaagucaaa aaugaagagg uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 397

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G2

<400> 397

gaauauguca cuugaacuca acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 398

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H2

<400> 398

ucacuugaac ucaacucaaa acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 399

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A3

<400> 399

ucaaaacuug aaagccuccu agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 400

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B3

<400> 400

caaaacuuga aagccuccua gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 401

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C3

<400> 401

aaaacuugaa agccuccuag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 402

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D3

<400> 402

aaacuugaaa gccuccuaga agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 403

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E3

<400> 403

aacuugaaag ccuccuagaa gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 404

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F3

<400> 404

guucuggagu uucagguuga uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 405

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G3

<400> 405

cacugguuug cagcgauaga ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 406

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H3

<400> 406

acugguuugc agcgauagau caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 407

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A4

<400> 407

cgauagauca uaaaaagacu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 408

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B4

<400> 408

cccaacugaa ggaggccauu ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 409

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C4

<400> 409

ccaacugaag gaggccauug gcguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 410

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D4

<400> 410

cuugauuuug gcucuggaga uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 411

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E4

<400> 411

uuuuggcucu ggagauagag aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 412

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F4

<400> 412

ucuggagaua gagaaucaaa ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 413

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G4

<400> 413

gaauugucuu gaucaauucu ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 414

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H4

<400> 414

aauugucuug aucaauucug gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 415

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A5

<400> 415

ggaggaaaua acuagaggaa caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 416

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B5

<400> 416

gaggaaauaa cuagaggaac aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 417

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C5

<400> 417

acucucuaua uccagacuuu ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 418

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D5

<400> 418

cucucuauau ccagacuuuu guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 419

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E5

<400> 419

ucucuauauc cagacuuuug uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 420

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F5

<400> 420

aacaauuaaa ccaacagcau agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 421

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G5

<400> 421

auuaaaccaa cagcauaguc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 422

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H5

<400> 422

aaccaacagc auagucaaau aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 423

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A6

<400> 423

accaacagca uagucaaaua aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 424

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B6

<400> 424

gaugcuauua ucuuguuuuu cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 425

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C6

<400> 425

aggacuagua uucaagaacc caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 426

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D6

<400> 426

ggacuaguau ucaagaaccc acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 427

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E6

<400> 427

aagaacuacu cccuuucuuc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 428

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F6

<400> 428

acuacucccu uucuucaguu gaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 429

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G6

<400> 429

cuacucccuu ucuucaguug aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 430

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H6

<400> 430

ccuuucuuca guugaaugaa auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 431

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A7

<400> 431

ggugcucuug gcuuggaaga uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 432

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B7

<400> 432

gugcucuugg cuuggaagau agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 433

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C7

<400> 433

auagagaaau uucugugggu ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 434

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D7

<400> 434

gaauacuagu ccuucugagc ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 435

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E7

<400> 435

uuauugauuc uaggcauucc ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 436

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F7

<400> 436

gucuacugug auguuauauc agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 437

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G7

<400> 437

cugauauaac aucacaguag acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 438

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H7

<400> 438

ugauauaaca ucacaguaga caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 439

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A8

<400> 439

gauauaacau cacaguagac auguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 440

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B8

<400> 440

cacuuguaug uucaccucug uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 441

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C8

<400> 441

uauaaauggu gguacauuca gcguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 442

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D8

<400> 442

ugguacauuc agcaggaaug ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 443

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E8

<400> 443

guccauggac auuaauucaa caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 444

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F8

<400> 444

uucaacaucg aauagaugga ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 445

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G8

<400> 445

auagauggau cacaaaacuu caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 446

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H8

<400> 446

uucaaugaaa cgugggagaa cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 447

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 A9

<400> 447

aguccccuua ccaucaagcc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 448

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 B9

<400> 448

uuugugaucc aucuauucga ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 449

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 C9

<400> 449

ugaauuaaug uccauggacu acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 450

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 D9

<400> 450

uuuacgaauu gaguuggaag acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 451

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 E9

<400> 451

ggcaaugucc ccaaugcaau ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 452

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 F9

<400> 452

gcaauguccc caaugcaauc ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 453

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 G9

<400> 453

guuuucuacu ugggaucaca aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 454

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3 H9

<400> 454

ccuuuugcuu ugugauccca agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 455

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3A 10

<400> 455

cuuuugcuuu gugaucccaa guguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 456

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3B 10

<400> 456

uugugauccc aaguagaaaa caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 457

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3C 10

<400> 457

aguugguuuc gugauuuccc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 458

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3D 10

<400> 458

guugguuucg ugauuuccca agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 459

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3E 10

<400> 459

guuucgugau uucccaagua aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 460

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3F 10

<400> 460

uuccagucuu ccaacucaau ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 461

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3G 10

<400> 461

aguauaucuu cucuaggccc aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 462

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3H 10

<400> 462

guauaucuuc ucuaggccca acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 463

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3A 11

<400> 463

ucuaggccca accaaaauuc ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 464

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3B 11

<400> 464

cuaggcccaa ccaaaauucu ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 465

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3C 11

<400> 465

gcccaaccaa aauucuccug aaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 466

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3D 11

<400> 466

uggugguggc augaugagug ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 467

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3E 11

<400> 467

ggugguggca ugaugagugu ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 468

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3F 11

<400> 468

ugaugagugu ggagaaaaca acguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 469

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3G 11

<400> 469

uguggagaaa acaaccuaaa ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 470

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3H 11

<400> 470

gguaaauaua acaaaccaag agguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 471

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3A 12

<400> 471

gaagaggauu aucuuggaag ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 472

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3B 12

<400> 472

aagaggauua ucuuggaagu cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 473

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3C 12

<400> 473

ucaaaaugga agguuauacu cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 474

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3D 12

<400> 474

caaaauggaa gguuauacuc uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 475

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3E 12

<400> 475

auguugaucc auccaacaga uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 476

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3F 12

<400> 476

cauccaacag auucagaaag cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 477

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting ANGPTL3

<220>

<223> description: MG3-6/3-4 ANGPTL3G 12

<400> 477

gccucaguuc auucaaagcu uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 478

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A1

<400> 478

ttgttcctct agttatttcc tc 22

<210> 479

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B1

<400> 479

atttgattct ctatctccag ag 22

<210> 480

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C1

<400> 480

tttgattctc tatctccaga gc 22

<210> 481

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D1

<400> 481

aagatttgct atgttagacg at 22

<210> 482

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E1

<400> 482

agatttgcta tgttagacga tg 22

<210> 483

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F1

<400> 483

gatttgctat gttagacgat gt 22

<210> 484

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G1

<400> 484

actttgtcca taagacgaag gg 22

<210> 485

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H1

<400> 485

agggccaaat taatgacata tt 22

<210> 486

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A2

<400> 486

gggccaaatt aatgacatat tt 22

<210> 487

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B2

<400> 487

tatgatctat cgctgcaaac ca 22

<210> 488

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C2

<400> 488

atgatctatc gctgcaaacc ag 22

<210> 489

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D2

<400> 489

caaaccagtg aaatcaaaga ag 22

<210> 490

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E2

<400> 490

aaaccagtga aatcaaagaa ga 22

<210> 491

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F2

<400> 491

acaagtcaaa aatgaagagg ta 22

<210> 492

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G2

<400> 492

gaatatgtca cttgaactca ac 22

<210> 493

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H2

<400> 493

tcacttgaac tcaactcaaa ac 22

<210> 494

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A3

<400> 494

tcaaaacttg aaagcctcct ag 22

<210> 495

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B3

<400> 495

caaaacttga aagcctccta ga 22

<210> 496

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C3

<400> 496

aaaacttgaa agcctcctag aa 22

<210> 497

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D3

<400> 497

aaacttgaaa gcctcctaga ag 22

<210> 498

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E3

<400> 498

aacttgaaag cctcctagaa ga 22

<210> 499

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F3

<400> 499

gttctggagt ttcaggttga tt 22

<210> 500

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G3

<400> 500

cactggtttg cagcgataga tc 22

<210> 501

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H3

<400> 501

actggtttgc agcgatagat ca 22

<210> 502

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A4

<400> 502

cgatagatca taaaaagact ga 22

<210> 503

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B4

<400> 503

cccaactgaa ggaggccatt gg 22

<210> 504

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C4

<400> 504

ccaactgaag gaggccattg gc 22

<210> 505

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D4

<400> 505

cttgattttg gctctggaga ta 22

<210> 506

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E4

<400> 506

ttttggctct ggagatagag aa 22

<210> 507

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F4

<400> 507

tctggagata gagaatcaaa tg 22

<210> 508

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G4

<400> 508

gaattgtctt gatcaattct gg 22

<210> 509

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H4

<400> 509

aattgtcttg atcaattctg ga 22

<210> 510

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A5

<400> 510

ggaggaaata actagaggaa ca 22

<210> 511

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B5

<400> 511

gaggaaataa ctagaggaac aa 22

<210> 512

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C5

<400> 512

actctctata tccagacttt tg 22

<210> 513

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D5

<400> 513

ctctctatat ccagactttt gt 22

<210> 514

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E5

<400> 514

tctctatatc cagacttttg ta 22

<210> 515

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F5

<400> 515

aacaattaaa ccaacagcat ag 22

<210> 516

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G5

<400> 516

attaaaccaa cagcatagtc aa 22

<210> 517

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H5

<400> 517

aaccaacagc atagtcaaat aa 22

<210> 518

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A6

<400> 518

accaacagca tagtcaaata aa 22

<210> 519

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B6

<400> 519

gatgctatta tcttgttttt ct 22

<210> 520

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C6

<400> 520

aggactagta ttcaagaacc ca 22

<210> 521

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D6

<400> 521

ggactagtat tcaagaaccc ac 22

<210> 522

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E6

<400> 522

aagaactact ccctttcttc ag 22

<210> 523

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F6

<400> 523

actactccct ttcttcagtt ga 22

<210> 524

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G6

<400> 524

ctactccctt tcttcagttg aa 22

<210> 525

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H6

<400> 525

cctttcttca gttgaatgaa at 22

<210> 526

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A7

<400> 526

ggtgctcttg gcttggaaga ta 22

<210> 527

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B7

<400> 527

gtgctcttgg cttggaagat ag 22

<210> 528

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C7

<400> 528

atagagaaat ttctgtgggt tc 22

<210> 529

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D7

<400> 529

gaatactagt ccttctgagc tg 22

<210> 530

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E7

<400> 530

ttattgattc taggcattcc tg 22

<210> 531

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F7

<400> 531

gtctactgtg atgttatatc ag 22

<210> 532

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G7

<400> 532

ctgatataac atcacagtag ac 22

<210> 533

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H7

<400> 533

tgatataaca tcacagtaga ca 22

<210> 534

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A8

<400> 534

gatataacat cacagtagac at 22

<210> 535

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B8

<400> 535

cacttgtatg ttcacctctg tt 22

<210> 536

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C8

<400> 536

tataaatggt ggtacattca gc 22

<210> 537

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D8

<400> 537

tggtacattc agcaggaatg cc 22

<210> 538

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E8

<400> 538

gtccatggac attaattcaa ca 22

<210> 539

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F8

<400> 539

ttcaacatcg aatagatgga tc 22

<210> 540

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G8

<400> 540

atagatggat cacaaaactt ca 22

<210> 541

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H8

<400> 541

ttcaatgaaa cgtgggagaa ct 22

<210> 542

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 A9

<400> 542

agtcccctta ccatcaagcc tc 22

<210> 543

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 B9

<400> 543

tttgtgatcc atctattcga tg 22

<210> 544

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 C9

<400> 544

tgaattaatg tccatggact ac 22

<210> 545

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 D9

<400> 545

tttacgaatt gagttggaag ac 22

<210> 546

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 E9

<400> 546

ggcaatgtcc ccaatgcaat cc 22

<210> 547

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 F9

<400> 547

gcaatgtccc caatgcaatc cc 22

<210> 548

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 G9

<400> 548

gttttctact tgggatcaca aa 22

<210> 549

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3 H9

<400> 549

ccttttgctt tgtgatccca ag 22

<210> 550

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3A 10

<400> 550

cttttgcttt gtgatcccaa gt 22

<210> 551

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3B 10

<400> 551

ttgtgatccc aagtagaaaa ca 22

<210> 552

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3C 10

<400> 552

agttggtttc gtgatttccc aa 22

<210> 553

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3D 10

<400> 553

gttggtttcg tgatttccca ag 22

<210> 554

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3E 10

<400> 554

gtttcgtgat ttcccaagta aa 22

<210> 555

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3F 10

<400> 555

ttccagtctt ccaactcaat tc 22

<210> 556

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3G 10

<400> 556

agtatatctt ctctaggccc aa 22

<210> 557

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3H 10

<400> 557

gtatatcttc tctaggccca ac 22

<210> 558

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3A 11

<400> 558

tctaggccca accaaaattc tc 22

<210> 559

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3B 11

<400> 559

ctaggcccaa ccaaaattct cc 22

<210> 560

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3C 11

<400> 560

gcccaaccaa aattctcctg aa 22

<210> 561

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3D 11

<400> 561

tggtggtggc atgatgagtg tg 22

<210> 562

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3E 11

<400> 562

ggtggtggca tgatgagtgt gg 22

<210> 563

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3F 11

<400> 563

tgatgagtgt ggagaaaaca ac 22

<210> 564

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3G 11

<400> 564

tgtggagaaa acaacctaaa tg 22

<210> 565

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3H 11

<400> 565

ggtaaatata acaaaccaag ag 22

<210> 566

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3A 12

<400> 566

gaagaggatt atcttggaag tc 22

<210> 567

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3B 12

<400> 567

aagaggatta tcttggaagt ct 22

<210> 568

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3C 12

<400> 568

tcaaaatgga aggttatact ct 22

<210> 569

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3D 12

<400> 569

caaaatggaa ggttatactc ta 22

<210> 570

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3E 12

<400> 570

atgttgatcc atccaacaga tt 22

<210> 571

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3F 12

<400> 571

catccaacag attcagaaag ct 22

<210> 572

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of ANGPTL3 target site

<220>

<223> description: MG3-6/3-4 ANGPTL3G 12

<400> 572

gcctcagttc attcaaagct tt 22

<210> 573

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 A1

<400> 573

accccuccac gguaccgggc ggguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 574

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 B1

<400> 574

accagcauac agagugacca ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 575

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 C1

<400> 575

ccagcauaca gagugaccac cgguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 576

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 D1

<400> 576

cagggucaug gucaccgacu ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 577

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 E1

<400> 577

ccucccaggc cuggaguuua uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 578

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 F1

<400> 578

cucccaggcc uggaguuuau ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 579

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 G1

<400> 579

caggcuggac cagcuggcuu uuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 580

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 H1

<400> 580

gguggcccca acugugauga ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 581

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 A2

<400> 581

gccccgccgc uucccacucc ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 582

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 B2

<400> 582

agugugcuga ccauacaguc cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 583

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 C2

<400> 583

ccugcaaaac agcugccaac cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 584

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 D2

<400> 584

cugcaaaaca gcugccaacc ugguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 585

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 E2

<400> 585

aauggcguag acacccucac ccguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 586

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 F2

<400> 586

uccugcugcc augccccagg ucguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 587

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting PCSK9

<220>

<223> description: MG3-6/3-4 PCSK9 G2

<400> 587

uggaaugcaa agucaaggag caguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 588

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 A1

<400> 588

acccctccac ggtaccgggc gg 22

<210> 589

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 B1

<400> 589

accagcatac agagtgacca cc 22

<210> 590

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 C1

<400> 590

ccagcataca gagtgaccac cg 22

<210> 591

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 D1

<400> 591

cagggtcatg gtcaccgact tc 22

<210> 592

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 E1

<400> 592

cctcccaggc ctggagttta tt 22

<210> 593

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 F1

<400> 593

ctcccaggcc tggagtttat tc 22

<210> 594

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 G1

<400> 594

caggctggac cagctggctt tt 22

<210> 595

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 H1

<400> 595

ggtggcccca actgtgatga cc 22

<210> 596

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 A2

<400> 596

gccccgccgc ttcccactcc tg 22

<210> 597

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 B2

<400> 597

agtgtgctga ccatacagtc ct 22

<210> 598

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 C2

<400> 598

cctgcaaaac agctgccaac ct 22

<210> 599

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 D2

<400> 599

ctgcaaaaca gctgccaacc tg 22

<210> 600

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 E2

<400> 600

aatggcgtag acaccctcac cc 22

<210> 601

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 F2

<400> 601

tcctgctgcc atgccccagg tc 22

<210> 602

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic oligonucleotides

<220>

<223> category: DNA sequence of PSCK9 target site

<220>

<223> description: MG3-6/3-4 PCSK9 G2

<400> 602

tggaatgcaa agtcaaggag ca 22

<210> 603

<211> 3705

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 coding sequence

<220>

<223> description: DNA sequence of MG3-6/3-4 coding sequence

<400> 603

atgcatgcgc ggccgcaagc tttaatacga ctcactataa ggaaaagcca gctccagcag 60

gcgctgctca ctcctcccca tcctctccct ctgtccctct gtccctctga ccctgcactg 120

tcccagcacc atggccccca agaagaagcg gaaagttggc ggcggaggca gcagcaccga 180

catgaagaac taccggatcg gcgtggacgt gggcgataga tctgttggac tggccgccat 240

cgagttcgac gatgatggac tgcccatcca gaagctggcc ctggtcacct ttagacacga 300

tggcggactg gaccccacca agaacaagac ccctatgagc cggaaagaga cacggggaat 360

cgccagacgg accatgcgga tgaacagaga gcggaagcgg cggctgagaa acctggacaa 420

cgtgctggaa aacctgggct actctgtgcc tgagggccct gagcctgaga catatgaggc 480

ctggacaagc agagccctgc tggcctctat caaactggcc tctgccgacg agctgaacga 540

acaccttgtc agagccgtgc ggcacatggc cagacataga ggatgggcca atccttggtg 600

gtccctggac cagctggaaa aggccagcca agagcctagc gagacattcg agatcatcct 660

ggccagagcc agagagctgt tcggcgagaa ggtgcccgct aatcctacac tgggaatgct 720

gggagccctg gccgctaaca atgaggtgct gctgaggccc agggacgaga agaagagaaa 780

gaccggatac gtgcggggca cccctctgat gtttgctcaa gttcgacagg gcgatcagct 840

ggccgagctg cggagaattt gtgaagtgca gggcatcgag gaccagtacg aggctctgag 900

actgggcgtg ttcgaccaca agcaccccta cgtgcccaaa gaaagagtgg gcaaagaccc 960

tctgaacccc agcaccaaca gaaccatcag agccagcctg gaatttcaag agttccgcat 1020

cctggacagc gtggccaatc tgagagtgcg gatcggcagc agagccaaga gggaactgac 1080

agaggccgag tatgatgccg ccgtggaatt cctgatggac tacgccgaca aagagcagcc 1140

tagctgggcc gatgtggccg agaaaattgg cgtgcccggc aacagactgg tggcccctgt 1200

tctggaagat gtgcagcaga aaacagcccc ttacgacaga agcagcgccg cctttgagaa 1260

ggccatgggc aagaaaaccg aggccagaca gtggtgggag tccaccgatg atgaccagct 1320

gagaagcctg ctgattgcct tcctggtgga cgccaccaac gacacagaag aagccgctgc 1380

tgaagccggc ctgagcgagc tgtataagtc ttggcctgcc gaggaaagag aggccctgtc 1440

caacatcgac ttcgagaagg gcagagtggc ctacagccaa gaaaccctga gcaagctgag 1500

cgagtacatg cacgagtaca gagtgggact gcacgaggct agaaaggccg tgttcggagt 1560

ggatgatacc tggcggcctc ctctggataa gctggaagaa cctacaggac agcctgccgt 1620

ggacagagtg ctgaccatcc tgagaagatt cgtgctggac tgcgagcggc aatggggcag 1680

acctagagcc atcaccgtgg aacacacacg gacaggcctg atgggcccaa cacagagaca 1740

gaagatcctg aacgagcaga agaagaaccg ggccgacaac gagagaatcc gggatgagct 1800

gagagaatct ggcgtggaca acccctccag agccgaagtt cggagacacc tgatcgtgca 1860

agagcaagag tgccagtgcc tgtactgcgg caccatgatc accaccacca caagcgagct 1920

ggaccacatc gttcctagag ccggtggcgg cagcagcaga agggaaaatc tggccgctgt 1980

gtgcagagcc tgcaacgcca agaagaaacg cgagctgttc tacgcctggg ctggcccagt 2040

gaagtcccaa gagacaatcg agagagtcag acagctgaag gcctttaagg acagcaagaa 2100

agccaagatg ttcaagaacc agatccgccg gctgaaccag accgaggccg atgagcctat 2160

cgacgaaaga agcctggcca gcacatctta cgccgctgtg gccgttagag agcggctgga 2220

acagcacttc aacgaaggcc tggcactgga cgacaagtcc agagtggtgc tggatgtgta 2280

tgccggcgct gtgaccagag agtctcgtag agctggcggc atcgacgagc ggattctgct 2340

gagaggcgag cgggacaaga acagattcga tgtgcggcat cacgccgtgg acgctgctgt 2400

tatgaccctg ctgaacagat ccgtggctct gaccctggaa cagagatcac agctgaggcg 2460

ggccttctac gagctggaac tggacaaact ggaccgggac cagctcaagc ctggcgagga 2520

ttggagaaac ttcaccggcc tgtacgaggc ctctcagaac aagttcagcg agtggaagaa 2580

agccgccaca gtgctgggag atctgctggc tgaagccatc gaggatgacg ccattgccgt 2640

ggtgtctcca ctgagactga ggccccagaa tggcagcgtg cacgacgata ccatcaacgc 2700

cgtgaagaag ctgacactgg gctctgcctg gcctgcagac gctgtgaaga gaatcgtgga 2760

ccccgagatc tacctggcta tgaaggacgt gctgggcaag ctgaaagagc tgcccgagga 2820

ttctgccaga tctctggaac tgtccgacgg ccggtacatc gaagccgatg acgaggtgct 2880

gttcttccca aagaaggccg ctagcatcct gacacctaga ggcgccgctg agatcggcaa 2940

ctctatccac catgccagac tgtatagctg gctgaccaag aagggcgagc tgaagtttgg 3000

catgctgaga gtgtacggcg ccgagtttcc ctggctgatg agagagtctg gaagccgcga 3060

cgtgctgcat atgcctattc accctggcag ccagagcttc agaggcatgc aggatggcgt 3120

gcggaaagcc gtggaaagcg gagaggctgt ggaattcggc tggatcaccc aggacgatga 3180

gctggaattc gaccccgagg actacattgc ccacggcgga gatgacgaac tgaacagact 3240

gctgcgagtg atgcccgaga gaaggtggcg agtggacggc ttctataacg ccggcacact 3300

gagaatcaga cccgctctgc tgtctgctga gcagctgcct tctgagctgc agaaaaaggt 3360

ggccgacaag accctgagcg acgtggaact gatcctgctg agggctgttc agcggggact 3420

gttcgtggcc atcagcagct ttctgcccct ggaaagcctg aaagtgatcc ggcggaacaa 3480

tctgggcttc cccaggtggc gcggaaacgg aaatctgccc accagctttg aagtgcggag 3540

cagcgctctg agagccctgg gagttgaagg atctggcgga aaaagacctg ccgccacaaa 3600

gaaagccgga caggccaaga aaaagaagtg accacacccc cattccccca ctccagatag 3660

aacttcagtt atatctcacg tgtctggagt tggatccatg catgc 3705

<210> 604

<211> 1166

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence: synthetic polypeptides

<220>

<223> category: MG3-6/3-4 box coding sequence

<220>

<223> description: protein sequence (including NLS) of MG3-6/3-4 box coding sequence

<400> 604

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Ser Thr

1 5 10 15

Asp Met Lys Asn Tyr Arg Ile Gly Val Asp Val Gly Asp Arg Ser Val

20 25 30

Gly Leu Ala Ala Ile Glu Phe Asp Asp Asp Gly Leu Pro Ile Gln Lys

35 40 45

Leu Ala Leu Val Thr Phe Arg His Asp Gly Gly Leu Asp Pro Thr Lys

50 55 60

Asn Lys Thr Pro Met Ser Arg Lys Glu Thr Arg Gly Ile Ala Arg Arg

65 70 75 80

Thr Met Arg Met Asn Arg Glu Arg Lys Arg Arg Leu Arg Asn Leu Asp

85 90 95

Asn Val Leu Glu Asn Leu Gly Tyr Ser Val Pro Glu Gly Pro Glu Pro

100 105 110

Glu Thr Tyr Glu Ala Trp Thr Ser Arg Ala Leu Leu Ala Ser Ile Lys

115 120 125

Leu Ala Ser Ala Asp Glu Leu Asn Glu His Leu Val Arg Ala Val Arg

130 135 140

His Met Ala Arg His Arg Gly Trp Ala Asn Pro Trp Trp Ser Leu Asp

145 150 155 160

Gln Leu Glu Lys Ala Ser Gln Glu Pro Ser Glu Thr Phe Glu Ile Ile

165 170 175

Leu Ala Arg Ala Arg Glu Leu Phe Gly Glu Lys Val Pro Ala Asn Pro

180 185 190

Thr Leu Gly Met Leu Gly Ala Leu Ala Ala Asn Asn Glu Val Leu Leu

195 200 205

Arg Pro Arg Asp Glu Lys Lys Arg Lys Thr Gly Tyr Val Arg Gly Thr

210 215 220

Pro Leu Met Phe Ala Gln Val Arg Gln Gly Asp Gln Leu Ala Glu Leu

225 230 235 240

Arg Arg Ile Cys Glu Val Gln Gly Ile Glu Asp Gln Tyr Glu Ala Leu

245 250 255

Arg Leu Gly Val Phe Asp His Lys His Pro Tyr Val Pro Lys Glu Arg

260 265 270

Val Gly Lys Asp Pro Leu Asn Pro Ser Thr Asn Arg Thr Ile Arg Ala

275 280 285

Ser Leu Glu Phe Gln Glu Phe Arg Ile Leu Asp Ser Val Ala Asn Leu

290 295 300

Arg Val Arg Ile Gly Ser Arg Ala Lys Arg Glu Leu Thr Glu Ala Glu

305 310 315 320

Tyr Asp Ala Ala Val Glu Phe Leu Met Asp Tyr Ala Asp Lys Glu Gln

325 330 335

Pro Ser Trp Ala Asp Val Ala Glu Lys Ile Gly Val Pro Gly Asn Arg

340 345 350

Leu Val Ala Pro Val Leu Glu Asp Val Gln Gln Lys Thr Ala Pro Tyr

355 360 365

Asp Arg Ser Ser Ala Ala Phe Glu Lys Ala Met Gly Lys Lys Thr Glu

370 375 380

Ala Arg Gln Trp Trp Glu Ser Thr Asp Asp Asp Gln Leu Arg Ser Leu

385 390 395 400

Leu Ile Ala Phe Leu Val Asp Ala Thr Asn Asp Thr Glu Glu Ala Ala

405 410 415

Ala Glu Ala Gly Leu Ser Glu Leu Tyr Lys Ser Trp Pro Ala Glu Glu

420 425 430

Arg Glu Ala Leu Ser Asn Ile Asp Phe Glu Lys Gly Arg Val Ala Tyr

435 440 445

Ser Gln Glu Thr Leu Ser Lys Leu Ser Glu Tyr Met His Glu Tyr Arg

450 455 460

Val Gly Leu His Glu Ala Arg Lys Ala Val Phe Gly Val Asp Asp Thr

465 470 475 480

Trp Arg Pro Pro Leu Asp Lys Leu Glu Glu Pro Thr Gly Gln Pro Ala

485 490 495

Val Asp Arg Val Leu Thr Ile Leu Arg Arg Phe Val Leu Asp Cys Glu

500 505 510

Arg Gln Trp Gly Arg Pro Arg Ala Ile Thr Val Glu His Thr Arg Thr

515 520 525

Gly Leu Met Gly Pro Thr Gln Arg Gln Lys Ile Leu Asn Glu Gln Lys

530 535 540

Lys Asn Arg Ala Asp Asn Glu Arg Ile Arg Asp Glu Leu Arg Glu Ser

545 550 555 560

Gly Val Asp Asn Pro Ser Arg Ala Glu Val Arg Arg His Leu Ile Val

565 570 575

Gln Glu Gln Glu Cys Gln Cys Leu Tyr Cys Gly Thr Met Ile Thr Thr

580 585 590

Thr Thr Ser Glu Leu Asp His Ile Val Pro Arg Ala Gly Gly Gly Ser

595 600 605

Ser Arg Arg Glu Asn Leu Ala Ala Val Cys Arg Ala Cys Asn Ala Lys

610 615 620

Lys Lys Arg Glu Leu Phe Tyr Ala Trp Ala Gly Pro Val Lys Ser Gln

625 630 635 640

Glu Thr Ile Glu Arg Val Arg Gln Leu Lys Ala Phe Lys Asp Ser Lys

645 650 655

Lys Ala Lys Met Phe Lys Asn Gln Ile Arg Arg Leu Asn Gln Thr Glu

660 665 670

Ala Asp Glu Pro Ile Asp Glu Arg Ser Leu Ala Ser Thr Ser Tyr Ala

675 680 685

Ala Val Ala Val Arg Glu Arg Leu Glu Gln His Phe Asn Glu Gly Leu

690 695 700

Ala Leu Asp Asp Lys Ser Arg Val Val Leu Asp Val Tyr Ala Gly Ala

705 710 715 720

Val Thr Arg Glu Ser Arg Arg Ala Gly Gly Ile Asp Glu Arg Ile Leu

725 730 735

Leu Arg Gly Glu Arg Asp Lys Asn Arg Phe Asp Val Arg His His Ala

740 745 750

Val Asp Ala Ala Val Met Thr Leu Leu Asn Arg Ser Val Ala Leu Thr

755 760 765

Leu Glu Gln Arg Ser Gln Leu Arg Arg Ala Phe Tyr Glu Leu Glu Leu

770 775 780

Asp Lys Leu Asp Arg Asp Gln Leu Lys Pro Gly Glu Asp Trp Arg Asn

785 790 795 800

Phe Thr Gly Leu Tyr Glu Ala Ser Gln Asn Lys Phe Ser Glu Trp Lys

805 810 815

Lys Ala Ala Thr Val Leu Gly Asp Leu Leu Ala Glu Ala Ile Glu Asp

820 825 830

Asp Ala Ile Ala Val Val Ser Pro Leu Arg Leu Arg Pro Gln Asn Gly

835 840 845

Ser Val His Asp Asp Thr Ile Asn Ala Val Lys Lys Leu Thr Leu Gly

850 855 860

Ser Ala Trp Pro Ala Asp Ala Val Lys Arg Ile Val Asp Pro Glu Ile

865 870 875 880

Tyr Leu Ala Met Lys Asp Val Leu Gly Lys Leu Lys Glu Leu Pro Glu

885 890 895

Asp Ser Ala Arg Ser Leu Glu Leu Ser Asp Gly Arg Tyr Ile Glu Ala

900 905 910

Asp Asp Glu Val Leu Phe Phe Pro Lys Lys Ala Ala Ser Ile Leu Thr

915 920 925

Pro Arg Gly Ala Ala Glu Ile Gly Asn Ser Ile His His Ala Arg Leu

930 935 940

Tyr Ser Trp Leu Thr Lys Lys Gly Glu Leu Lys Phe Gly Met Leu Arg

945 950 955 960

Val Tyr Gly Ala Glu Phe Pro Trp Leu Met Arg Glu Ser Gly Ser Arg

965 970 975

Asp Val Leu His Met Pro Ile His Pro Gly Ser Gln Ser Phe Arg Gly

980 985 990

Met Gln Asp Gly Val Arg Lys Ala Val Glu Ser Gly Glu Ala Val Glu

995 1000 1005

Phe Gly Trp Ile Thr Gln Asp Asp Glu Leu Glu Phe Asp Pro Glu

1010 1015 1020

Asp Tyr Ile Ala His Gly Gly Asp Asp Glu Leu Asn Arg Leu Leu

1025 1030 1035

Arg Val Met Pro Glu Arg Arg Trp Arg Val Asp Gly Phe Tyr Asn

1040 1045 1050

Ala Gly Thr Leu Arg Ile Arg Pro Ala Leu Leu Ser Ala Glu Gln

1055 1060 1065

Leu Pro Ser Glu Leu Gln Lys Lys Val Ala Asp Lys Thr Leu Ser

1070 1075 1080

Asp Val Glu Leu Ile Leu Leu Arg Ala Val Gln Arg Gly Leu Phe

1085 1090 1095

Val Ala Ile Ser Ser Phe Leu Pro Leu Glu Ser Leu Lys Val Ile

1100 1105 1110

Arg Arg Asn Asn Leu Gly Phe Pro Arg Trp Arg Gly Asn Gly Asn

1115 1120 1125

Leu Pro Thr Ser Phe Glu Val Arg Ser Ser Ala Leu Arg Ala Leu

1130 1135 1140

Gly Val Glu Gly Ser Gly Gly Lys Arg Pro Ala Ala Thr Lys Lys

1145 1150 1155

Ala Gly Gln Ala Lys Lys Lys Lys

1160 1165

<210> 605

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-7-1

<400> 605

gagcuggcca cugugcgagg uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 606

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-20-1

<400> 606

uucagcaagu ccacuguugu cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 607

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-7-35

<400> 607

gagcuggcca cugugcgagg uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 608

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-20-35

<400> 608

uucagcaagu ccacuguugu cuguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 609

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-7-42

<400> 609

gagcuggcca cugugcgagg uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

<210> 610

<211> 110

<212> RNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesis of polynucleotides

<220>

<223> category: MG3-6/3-4 sgRNA targeting mouse HAO-1

<220>

<223> description: mH364-7-45

<400> 610

gagcuggcca cugugcgagg uaguugagaa ucgaaagauu cuuaauaagg cauccuuccg 60

augcugacuu cucaccgucc guuuuccaau aggagcgggc gguauguuuu 110

Claims

1. A fusion endonuclease, comprising:

(a) An N-terminal sequence comprising at least a portion of a RuvC domain, REC domain, or HNH domain of an endonuclease having at least 55% sequence identity to SEQ ID No. 696 or a variant thereof; and

(b) A C-terminal sequence comprising a WED, TOPO or CTD domain of an endonuclease having at least 55% sequence identity to any one of SEQ ID NOs 697-721 or a variant thereof, wherein said N-terminal sequence and said C-terminal sequence are not naturally present together in the same reading frame.

2. The fusion endonuclease of claim 1, wherein the N-terminal sequence and the C-terminal sequence are derived from different organisms.

3. The fusion endonuclease of any one of claims 1 or 2, wherein the N-terminal sequence further comprises a RuvC-I, BH or RuvC-II domain.

4. A fusion endonuclease according to any one of claims 1 to 3, wherein the C-terminal sequence further comprises a PAM interaction domain.

5. The fusion endonuclease according to any one of claims 1 to 4, wherein said fusion endonuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs 1-27 or 108.

6. The fusion endonuclease according to any one of claims 1 to 5, wherein said fusion endonuclease is configured to bind to PAM (SEQ ID NO: 53) that is not nnRGGnT.

7. The fusion endonuclease of claim 6, wherein the fusion endonuclease is configured to bind PAM comprising any one of SEQ ID NOs 46-52 or 54-66.

8. An endonuclease comprising an engineered amino acid sequence having at least 55% sequence identity to any one of SEQ ID NOs 1-27 or 108 or a variant thereof.

9. An endonuclease comprising an engineered amino acid sequence having at least 55% sequence identity to any one of SEQ ID NOs 109-110 or a variant thereof.

10. An engineered nuclease system, comprising:

(a) An endonuclease according to any one of claims 1 to 9; and

(b) An engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the engineered guide ribonucleic acid structure comprising:

a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence; wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease.

11. The engineered nuclease system of claim 10, wherein the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease.

12. The engineered nuclease system of claim 10 or 11, wherein the endonuclease is derived from an uncultured microorganism.

13. The engineered nuclease system of any one of claims 10-12, wherein the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12 c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease.

14. The engineered nuclease system of any one of claims 10-13, wherein the endonuclease has less than 86% identity to a SpyCas9 endonuclease.

15. The engineered nuclease system of any one of claims 10-14, wherein the system further comprises Mg ²⁺ Is a source of (a).

16. The engineered nuclease system of any one of claims 10-15, wherein the endonuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs 8-12, 26-27 or 108, or a variant thereof.

17. The engineered nuclease system of any one of claims 10-16, wherein the guide ribonucleic acid sequence comprises a sequence having at least 80% identity to a non-degenerate nucleotide of any one of SEQ ID NOs 33, 34, 44, 45, 78, 84, or 87.

18. An engineered nuclease, comprising:

(a) A class II, type II Cas enzyme RuvC and HNH domain, or variants thereof, having at least 55% sequence identity to the RuvC and HNH domain of any one of SEQ ID NOs 1-27, 108 or 109-110; and

(b) A class II, type II Cas enzyme PAM Interaction (PI) domain or variant thereof, the class II, type II Cas enzyme PI domain having at least 55% sequence identity to a PAM Interaction (PI) domain of any one of SEQ ID NOs 1-27, 108 or 109-110.

19. The engineered nuclease of claim 18, wherein (a) and (b) do not naturally occur together.

20. The engineered nuclease of claim 18 or 19, wherein the class II, type II Cas enzyme is derived from an uncultured microorganism.

21. The engineered nuclease of any one of claims 18-20, wherein the endonuclease has less than 86% identity to a SpyCas9 endonuclease.

22. The engineered nuclease of any one of claims 18-21, wherein the engineered nuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs 1-27.

23. An engineered nuclease system, comprising:

(a) An endonuclease according to any one of claims 18 to 22; and

i. a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and configured to bind to the endonuclease.

24. The engineered nuclease system of claim 23, wherein the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease.

25. The engineered nuclease system of claim 23 or 24, wherein the guide ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of any one of SEQ ID NOs 28-32 or 33-44, or a variant thereof.

26. The engineered nuclease system of any one of claims 23-25, further comprising a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site.

27. The engineered nuclease system of claim 26, wherein the PAM sequence is located 3' to the target deoxyribonucleic acid sequence.

28. The engineered nuclease system of any one of claims 26 to 27, wherein the PAM sequence comprises any one of SEQ ID NOs 46-66.

29. A method of targeting an albumin gene, the method comprising introducing the system according to any one of claims 23 to 28 into a cell, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence comprising any one of SEQ ID NOs 67-86.

30. A method of targeting HAO1 gene, the method comprising introducing the system of any one of claims 23 to 28 into a cell, wherein the guide ribonucleic acid sequence is configured to hybridize to any one of SEQ ID NOs 611-633.

31. The method of claim 30, wherein the guide ribonucleic acid sequence is configured to hybridize to any of SEQ ID NOs 615, 618, 620, 624, or 626.

32. The method of claim 30, wherein the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOs 645-684.

33. The method of claim 32, wherein the guide ribonucleic acid comprises a sequence having at least 80% identity to any one of SEQ ID NOs 645-649, 652-656, 660-671, 674-675, or 681-684 or a sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs 645-649, 652-656, 660-671, 674-675, or 681-684.

34. A method of disrupting a HAO-1 locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the HAO-1 locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: targeting sequences having at least 80% identity to SEQ ID NO 611-626 or 627-633.

35. The method of claim 34, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

36. The method of claim 34 or 35, wherein the class 2, type II Cas endonuclease comprises a sequence having at least 55% identity to SEQ ID No. 10 or a variant thereof.

37. The method of any one of claims 34 to 36, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

38. The method of any one of claims 34 to 36, wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 618, 620, 624 or 626 or a sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs 618, 620, 624 or 626.

39. The method of any one of claims 34 to 36, wherein the engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs of table 9 or table 12.

40. A method of disrupting a TRAC locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRAC locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO 139-158; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS 119-138.

41. The method of claim 40, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

42. The method of claim 40 or 41, wherein the class 2, type II Cas endonuclease comprises the fusion endonuclease having at least 55% identity to SEQ ID No. 10 or a variant thereof.

43. The method of any one of claims 40 to 42, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

44. The method of any one of claims 40 to 42, wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 121, 132, 136, 130, 134, 135 or 137 or a sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs 121, 132, 136, 130, 134, 135 or 137.

45. The method of any one of claims 40 to 42, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7A.

46. A method of disrupting a B2M locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the B2M locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO. 185-210; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS 159-184.

47. The method of claim 46, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

48. The method of claim 46 or 47, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55% identity to SEQ ID No. 10 or a variant thereof.

49. The method of any one of claims 46 to 48, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

50. The method of any one of claims 46 to 48, wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 159, 165, 168, 174 or 184 or a sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs 159, 165, 168, 174 or 184.

51. The method of any one of claims 46 to 48, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7B.

52. A method of disrupting a TRBC1 locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC1 locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO. 252-292; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 211-251.

53. The method of claim 52, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

54. The method of claim 52 or 53, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55% identity to SEQ ID No. 10 or a variant thereof.

55. The method of any one of claims 52 to 54, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

56. The method of any one of claims 52 to 54, wherein the engineered guide RNA is a targeting sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs 211, 212, 215, 241 or 242 or comprising a targeting sequence having at least 80% identity to any one of SEQ ID NOs 211, 212, 215, 241 or 242.

57. The method of any one of claims 52 to 54, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7C.

58. A method of disrupting a TRBC2 locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC2 locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO 338-382; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS.293-337.

59. The method of claim 58, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

60. The method of claim 58 or 59, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55% identity to SEQ ID No. 10 or a variant thereof.

61. The method of any one of claims 58 to 60, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

62. The method of any one of claims 58 to 61, wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 296, 306 or 332 or comprises a targeting sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs 296, 306 or 332.

63. The method of any one of claims 58 to 61, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7C.

64. A method of disrupting an ANGPTL3 locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the ANGPTL3 locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO 478-572; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 383-477.

65. The method of claim 64, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

66. The method of claim 64 or 65, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55% identity to SEQ ID No. 10 or a variant thereof.

67. The method of any one of claims 64 to 66, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

68. The method of any one of claims 64 to 66, wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 419, 425, 431, 439, 447, 453, 461, 467, 471 or 473 or a sequence having at least 80% identity to any one of SEQ ID NOs 419, 425, 431, 439, 447, 453, 461, 467, 471 or 473.

69. The method of any one of claims 64 to 66, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7D.

70. A method of disrupting a PCSK9 locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the PCSK9 locus,

wherein the engineered guide RNA is configured to hybridize to or comprise: a targeting sequence having at least 80% identity to SEQ ID NO 588-602; or (b)

Wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs 573-587.

71. The method of claim 70, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

72. The method of claim 70 or 71, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55% identity to SEQ ID No. 10 or a variant thereof.

73. The method of any one of claims 70-72, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

74. The method of any one of claims 70-73, wherein the engineering guide comprises a sequence having at least 80% identity to any one of SEQ ID NOs 574, 578, 581, or 585.

75. The method of any one of claims 70 to 73, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 7E.

76. A method of disrupting an albumin locus in a cell, the method comprising introducing into the cell:

(a) Class 2, type II Cas endonuclease; and

(b) An engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the albumin locus,

wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 67-86 or 646-695 or wherein the engineered guide RNA comprises a targeting sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOS: 67-86 or 646-695.

77. The method of claim 76, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease according to any one of claims 1 to 9 or 18 to 22.

78. The method of claim 76 or 77, wherein the class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55% identity to SEQ ID No. 10 or a variant thereof.

79. The method of any one of claims 76 to 78, wherein the engineered guide RNA comprises a sequence having at least 80% sequence identity to a non-degenerate nucleotide of SEQ ID No. 722.

80. The method of any one of claims 76 to 79, wherein the engineered guide RNA is complementary to or comprises: a sequence having at least 80% identity to any one of SEQ ID NOs 67, 68, 70, 71, 72, 76, 79, 80, 647, 648, 649, 653, 654, 655, 656, 673, 680, 681 or 682.

81. The method of any one of claims 76 to 79, wherein the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs of table 6.