KR20220145438A - An engineered guide RNA for the optimized CRISPR/Cas12f1 system and use thereof - Google Patents

Abstract

In the present invention, an engineered Cas12f1 guide RNA for increasing an intracellular gene editing activity of a CRISPR/Cas12f1 system is provided by overcoming limitations of a prior art. The engineered Cas12f1 guide RNA is a modification of a part of a structure of a guide RNA found in nature. In the engineered Cas12f1 guide RNA, at least a part of a scaffold region having a role to interact with Cas12f1 protein is modified. The engineered scaffold region is different from a scaffold region of the guide RNA found in nature. The engineered guide RNA comprises the engineered scaffold region and a spacer.

Description

An engineered guide RNA for the optimized CRISPR/Cas12f1 system and use thereof

Disclosed herein is a technology for the field of using the CRISPR/Cas system, particularly the CRISPR/Cas12f1 system, for gene editing.

The CRISPR/Cas12f1 system is a CRISPR/Cas system classified as Class 2, Type V. In a previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), the CRISPR/Cas14 system, a CRISPR/Cas system derived from archaebacteria for the first time, was revealed. Then, in a subsequent study (Karvelis et al., Nucleic Acids Research, Vol. 48, No. 9 5017 (2020)), the CRISPR/Cas14 system was classified as a CRISPR/Cas12f system. The CRISPR/Cas12f1 system belongs to the V-F1 system, which is a subtype of one of the CRISPR/Cas systems classified into Class 2 and Type V, and includes the CRISPR/Cas14a system having Cas14a as an effector protein. The CRISPR/Cas12f1 system is characterized in that the size of the effector protein is significantly smaller than that of the CRISPR/Cas9 system. However, as revealed in previous studies (Harington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018), US 2020/0190494 A1), the CRISPR/Cas12f1 system, in particular CRISPR/ The Cas14a system has the ability to cut single-stranded DNA, but has no or extremely low cleavage activity for double-stranded DNA, limiting its application to gene editing technology.

As a continuing study, in the recent prior literature (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)), Cas12f1 protein is a dimer in the CRISPR / Cas12f1 system. , and the structure of guide RNAs. It was found by the above literature that there is a so-called disordered region that does not directly interact with the Cas12f1 protein in the guide RNA portion, and another prior literature (Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, In bioRxiv (2020)), the disordered region was removed and double-stranded DNA cleavage efficiency was examined in vitro. However, the preceding studies do not 1) show or increase intracellular gene editing activity (eg, indel generation efficiency), and 2) there are experiments that remove the disordered region, but rather the cleavage activity is low. 3) Moreover, as the above experiment was conducted in vitro, it failed to reveal what modifications were made to show intracellular gene editing activity or to increase efficiency.

An object of the present specification is to provide an engineered Cas12f1 guide RNA that can be used in the CRISPR/Cas12f1 system to increase gene editing efficiency.

In the present specification, it is intended to provide an engineered scaffold region that can be included in the engineered Cas12f1 guide RNA to increase gene editing efficiency.

An object of the present specification is to provide an engineered CRISPR/Cas12f1 complex with increased gene editing efficiency.

An object of the present specification is to provide an engineered CRISPR/Cas12f1 system with increased gene editing efficiency.

An object of the present specification is to provide a vector having a nucleic acid sequence encoding each component of the engineered CRISPR/Cas12f1 system.

An object of the present specification is to provide a gene editing method using the engineered CRISPR/Cas12f1 system.

In the present specification, it is intended to provide the use of the engineered CRISPR/Cas12f1 system.

In order to solve the above problem, herein disclosed is an engineered guide RNA for the CRISPR/Cas12f1 system, comprising:

engineered scaffold area; and

spacer;

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and

5'-AUGCAAC-3'.

In order to solve the above problem, herein disclosed is an engineered guide RNA for the CRISPR/Cas12f1 system, comprising:

engineered scaffold area; and

spacer; and

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUUCCUCUCCCAAAAUUCGAGAACCAAUGAAUGAAUUCGAUGAAGAAUGAAUGAAUGAUGAAGUGAGAAUGAAUGAAUGAUGAUGAGAAUGAAUGAAUGAUGAAUGAAGAAU characterized by that the sequence differs by the sequence

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) No. 117), 5'-AACAAAUUCAUUUUGAAACGAAUGAAGGA-3' (SEQ ID NO: 293), 5'-AACAAAUUCAUUUUUGAAAACGAAUGAAGGA-3' (SEQ ID NO: 294), 5'-AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAAU-GAAAAAGGAUGA (SEQ ID NO: 296), 5'-AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 297), 5'-AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 298), 5'-AACAAAUUCAUUCAUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAACUGAAUGAAUUUUUCCUCUGAAUGAAGAAUUCA 3' (SEQ ID NO: 300), 5'-AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 301), 5'-AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAGU-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAU AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 304), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 305), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 306), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 307), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 308 ), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 309), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 310), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 311), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열 312), 5'-AACAAAUUCAUUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 313), and 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO:314) selected from the group consisting of; and

5'-AUGCAAC-3'.

In order to solve the above problem, herein disclosed is an engineered guide RNA for the CRISPR/Cas12f1 system, comprising:

engineered scaffolds; and

spacer,

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer has a length of 10 to 30 nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

a first sequence represented by 5'-A-3';

a second sequence represented by 5'-CCGCUUCAC-3' (SEQ ID NO: 432);

a third sequence represented by 5'-UUAG-3';

a fourth sequence represented by 5'-AGUGAAGGUGG-3' (SEQ ID NO: 433);

a fifth sequence represented by 5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

a sixth sequence represented by 5'-AACAAA-3';

linker;

a seventh sequence represented by 5'-GGA-3'; and

The eighth sequence represented by 5'-AUGCAAC-3'.

In order to solve the above problem, herein disclosed is an engineered guide RNA for CRISPR/Cas12f1 system comprising:

engineered scaffold area; and

spacer;

In this case, the spacer has a length of 10 to 30 nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region comprises the following sequence:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), a sequence selected from the group consisting of 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9),

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order,

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11), and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), 5'-AACAAAUUCAU- an engineered tracrRNA to which a sequence selected from the group consisting of 3' (SEQ ID NO: 67), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), and 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 12) is linked; and

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', 5' -AAUGAAGGA-3', and a sequence selected from the group consisting of 5'-GAAUGAAGGA-3' (SEQ ID NO: 14), and

5'-AUGCAAC-3' linked engineered crRNA repeat sequence portion,

At this time, the 3' end of the engineered crRNA repeat sequence portion is connected to the 5' end of the spacer,

If the sequence of the engineered tracrRNA is identical to 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 1), except that the sequence is identical to that of the engineered tracrRNA repeats that the sequence is identical to 5'-CUUCACUGAUAAAGUGGAGAGAACCGCUUCACCAAAAGCUGUCCCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 1). characterized.

In order to solve the above problem, disclosed herein is an engineered CRISPR/Cas12f1 complex capable of editing a nucleic acid comprising a target sequence, comprising:

Cas12f1 protein; and

engineered guide RNA,

In this case, the engineered guide RNA comprises:

engineered scaffold area; and

spacer;

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and

5'-AUGCAAC-3'.

In order to solve the above problem, the present specification discloses a vector capable of expressing each component of the CRISPR/Cas12f1 system, comprising:

a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein;

a first promoter sequence operably linked to said first sequence;

a second sequence comprising a nucleic acid sequence encoding the engineered guide RNA; and

a second promoter sequence operably linked to said second sequence;

In this case, the engineered guide RNA comprises:

engineered scaffold area; and

spacer;

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and

5'-AUGCAAC-3'.

In order to solve the above problem, disclosed herein is a method for editing a nucleic acid comprising a target sequence in a cell, comprising:

delivering the Cas12f1 protein or a nucleic acid encoding the same, and an engineered guide RNA or a nucleic acid encoding the name into a cell;

Due to this, the CRISPR/Cas12f1 complex may be formed in the cell,

Due to this, the nucleic acid comprising the target sequence can be edited by the CRISPR/Cas12f1 complex,

The engineered guide RNA is characterized in that it comprises:

engineered scaffold area; and

spacer;

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and

5'-AUGCAAC-3'.

In order to solve the above problem, disclosed herein is a method for editing a nucleic acid comprising a target sequence in a cell, comprising:

delivering the Cas12f1 protein or a nucleic acid encoding the same, and an engineered guide RNA or a nucleic acid encoding the name into a cell;

Due to this, the CRISPR/Cas12f1 complex may be formed in the cell,

Due to this, the nucleic acid comprising the target sequence can be edited by the CRISPR/Cas12f1 complex,

The engineered guide RNA is characterized in that it comprises:

engineered scaffold area; and

spacer; and

At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,

The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,

The sequence of the engineered scaffold region is 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUUCCUCUCCCAAAAUUCGAGAACCAAUGAAUGAAUUCGAUGAAGAAUGAAUGAAUGAUGAAGUGAGAAUGAAUGAAUGAUGAUGAGAAUGAAUGAAUGAUGAAUGAAGAAU characterized by that the sequence differs by the sequence

The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) No. 117), 5'-AACAAAUUCAUUUUGAAACGAAUGAAGGA-3' (SEQ ID NO: 293), 5'-AACAAAUUCAUUUUUGAAAACGAAUGAAGGA-3' (SEQ ID NO: 294), 5'-AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAAU-GAAAAAGGAUGA (SEQ ID NO: 296), 5'-AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 297), 5'-AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 298), 5'-AACAAAUUCAUUCAUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAACUGAAUGAAUUUUUCCUCUGAAUGAAGAAUUCA 3' (SEQ ID NO: 300), 5'-AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 301), 5'-AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAGU-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAU AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 304), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 305), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 306), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 307), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 308 ), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 309), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 310), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 311), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열 312), 5'-AACAAAUUCAUUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 313), and 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO:314) selected from the group consisting of; and

5'-AUGCAAC-3'.

When the CRISPR/Cas12f1 system including the engineered Cas12f1 guide RNA having the engineered scaffold region provided herein is used for gene editing, high gene editing efficiency is achieved compared to when using the CRISPR/Cas12f1 system found in nature. indicates.

1 is a schematic diagram illustratively showing the engineered Cas12f1 guide RNA disclosed herein.
2 to 4 are graphs showing average indel efficiencies for Examples 1.1 to 1.13 targeting DY2 among the examples disclosed in Experimental Example 2. FIG. In this case, Ex is an abbreviation for Example, Comp is an abbreviation for Comparative Example, and Control is a negative control that is not treated with CRISPR/Cas12f1.
5 to 8 are graphs showing average indel efficiencies for Examples 2.1 to 2.13 targeting DY10 among the examples disclosed in Experimental Example 2. FIG. In this case, Ex is an abbreviation for Example, Comp is an abbreviation for Comparative Example, and Control is a negative control that is not treated with CRISPR/Cas12f1.
9 to 12 are graphs showing average indel efficiencies for Examples 3.1 to 3.13 targeting Intergenic-22 among the Examples disclosed in Experimental Example 2; In this case, Ex is an abbreviation for Example, Comp is an abbreviation for Comparative Example, and Control is a negative control that is not treated with CRISPR/Cas12f1.
13 shows the average indels for Examples 1.13 to Example 1.14 targeting DY2, Examples 2.13 to Example 2.14 targeting DY10, and Examples 3.13 to Example 3.14 targeting Intergenic-22 among the examples disclosed in Experimental Example 2; This is a graph showing the efficiency. In this case, Ex is an abbreviation for Example, Comp is an abbreviation for Comparative Example, and Control is a negative control that is not treated with CRISPR/Cas12f1.

Definition of Terms

approximately

As used herein, the term “about” refers to 30, 25, 20, 25, 10, 9, 8, 7 with respect to a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length. , means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 6, 5, 4, 3, 2 or 1%.

A,T,C,G, and U

As used herein, the symbols A, T, C, G and U are interpreted as meanings understood by those of ordinary skill in the art. It may be appropriately interpreted as a base, nucleoside or nucleotide on DNA or RNA according to context and technology. For example, when it means a base, it can be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U) itself, respectively, and when it means a nucleoside, It can be interpreted as adenosine (A), thymine (T), cytidine (C), guanosine (G) or uridine (U), respectively, and if it means a nucleotide in the sequence, it includes each of the nucleosides should be construed as meaning a nucleotide that

operably linked

The term "operably linked" as used herein, in gene expression technology, means that a specific component is linked to another component, such that the specific component is linked such that it functions in an intended manner. For example, when a promoter sequence is operatively linked with a coding sequence, it is meant that the promoter is linked so as to affect the transcription and/or expression of the coding sequence in a cell. In addition, the term includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

target gene or target nucleic acid

"Target gene" or "target nucleic acid" as used herein basically refers to a gene or nucleic acid in a cell to be subjected to gene editing. The target gene or target nucleic acid may be used interchangeably, and may refer to the same subject. The target gene or target nucleic acid may refer to both a unique gene or nucleic acid possessed by a target cell, or an externally-derived gene or nucleic acid, unless otherwise specified, and is not particularly limited as long as it can be subjected to gene editing. The target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. In addition, the term includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

target sequence

As used herein, “target sequence” refers to a specific sequence recognized by the CRISPR/Cas complex to cleave a target gene or target nucleic acid. The target sequence may be appropriately selected depending on the purpose. Specifically, "target sequence" refers to a sequence included in a target gene or target nucleic acid sequence, and refers to a sequence having complementarity with a spacer sequence included in a guide RNA provided herein or an engineered guide RNA. In general, the spacer sequence is determined in consideration of the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR/Cas system. The target sequence may refer only to a specific strand complementary to the guide RNA of the CRISPR/Cas complex, or may refer to the entire target double strand including the specific strand portion, which is appropriately interpreted according to the context. In addition, the term includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

vector

As used herein, unless otherwise specified, "vector" refers to any material capable of transporting genetic material into a cell. For example, a vector may be, but is not limited to, a DNA molecule comprising the genetic material of interest, for example, a nucleic acid encoding an effector protein of the CRISPR/Cas system, and/or a nucleic acid encoding a guide RNA. . The above terms include all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

found in nature

As used herein, the term "found in nature" means an unmodified object found in the natural world, and is used to distinguish it from an "engineered object" that has been artificially deformed. The "genes, nucleic acids, DNA, RNA, etc. found in nature" are used as a concept encompassing all genes, nucleic acids, DNA, and RNA in wild-type and mature form (active form). The term includes all other meanings recognized by those skilled in the art, and should be appropriately interpreted according to the context.

engineered

As used herein, the term "engineered" is a term used to distinguish substances, molecules, etc. having a constitution that already exist in nature, and means that artificial modifications are applied to the substances, molecules, etc. For example, in the case of "engineered guide RNA", it refers to a guide RNA in which an artificial change is applied to the composition of a guide RNA existing in nature. In addition, the term includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

NLS (Nuclear Localization Sequence, or Signal)

As used herein, the term “NLS” refers to a peptide of a certain length that acts as a kind of “tag” attached to a protein to be transported when a substance outside the cell nucleus is transported into the nucleus by nuclear transport, or its means sequence. Specifically, the NLS is the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 277); NLS from nucleoplasmin (eg, nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 278)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 279) or RQRRNELKRSP (SEQ ID NO: 280); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 281); sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 282) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 283) and PPKKARED (SEQ ID NO: 284) of the myoma T protein; the sequence PQPKKKPL of human p53 (SEQ ID NO: 285); sequence SALIKKKKKMAP of mouse c-abl IV (SEQ ID NO: 286); sequences DRLRR (SEQ ID NO: 287) and PKQKKRK (SEQ ID NO: 288) of influenza virus NS1; sequence RKLKKKIKKL (SEQ ID NO: 289) of hepatitis virus delta antigen; sequence REKKKFLKRR (SEQ ID NO: 290) of mouse Mx1 protein; sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 291) of human poly(ADP-ribose) polymerase; or an NLS sequence derived from the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 292) of a steroid hormone receptor (human) glucocorticoid. As used herein, the term “NLS” includes all meanings recognized by those of ordinary skill in the art, and may be appropriately interpreted according to the context.

NES (Nuclear Export Sequence, or Signal)

As used herein, the term "NES" refers to a peptide of a certain length that acts as a kind of "tag" attached to a protein to be transported when a material inside the cell nucleus is transported to the outside of the nucleus by nuclear transport, or its means sequence. As used herein, the term “NES” includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.

tag

As used herein, the term “tag” refers to a functional domain added to facilitate the tracking and/or separation and purification of peptides or proteins. Specifically, the tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag, and a tag protein such as a thioredoxin (Trx) tag, a green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), autofluorescent proteins such as HcRED, DsRed, and glutathione-S-transferase (GST), horseradish ( horseradish) peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, reporter genes such as luciferase, but are not limited thereto. As used herein, the term "tag" includes all meanings recognizable to those of ordinary skill in the art, and may be appropriately interpreted according to the context.

Background - Structure of the CRISPR/Cas12f1 complex

CRISPR/Cas12f system

The CRISPR/Cas12f system belongs to the V-F subtype of the type V CRISPR/Cas system, which is further divided into V-F1 to V-F3 variants. The CRISPR/Cas12f system is one of the effector proteins named Cas14 in a previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), Cas14a, Cas14b, and Cas14c variants. CRISPR/Cas14 systems comprising Among them, the CRISPR/Cas14a system including the Cas14a effector protein is classified as the CRISPR/Cas12f1 system (Makarova et al., Nature Reviews, Microbiology volume 18, 67 (2020)). Recent previous studies (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021), Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, bioRxiv (2020)), etc. have revealed the structure of the CRISPR/Cas12f1 complex, which will be briefly described below.

Structure of the CRISPR/Cas12f1 complex

Within the CRISPR/Cas12f1 complex, it was found that two molecules of the Cas12f1 protein bind to the guide RNA in the form of a dimer to form a complex. The Cas12f1 protein is divided into an amino-terminal domain (NTD) and a carboxy-terminal domin (CTD), and has a structure in which the two domains are connected through a linker loop. The NTD consists of wedge (WED), recognition (REC), and zinc finger (ZF) domains, and the CTD consists of another ZF domain and a RuvC domain. The structure of the Cas12f1 protein dimer can be largely divided into a REC lobe and a nuclease lobe. The REC lobe is composed of a WED domain, a ZF domain, and a REC domain of one Cas12f1 protein constituting a dimer and a WED domain, a ZF domain, and a REC domain of the other Cas12f1 protein. The nuclease lobe is composed of the RuvC domain and TNB domain of one Cas12f1 protein constituting the dimer, and the RuvC domain and TNB domain of the other Cas12f1 protein. All or part of each domain of the Cas12f1 protein recognizes a specific part of the scaffold region of the Cas12f1 guide RNA, respectively, and forms a CRISPR/Cas12f1 complex.

Structure of Cas12f1 guide RNA

Cas12f1 guide RNA can be largely divided into a spacer and a scaffold region, and the scaffold region is composed of five stems (named Stem 1 to Stem 5) and one pseudoknot (PK). The Cas12f1 guide RNA includes two structures in which a part of tracrRNA (tracrRNA anti-repeat) and a part of crRNA repeat (crRNA repeat) are complementary to form a duplex. Named as tracrRNA anti-repeat (R:AR) moiety. The Stem 5 (R:AR2), and PK (R:AR1) form this crRNA repeat-tracrRNA anti-repeat duplex structure. In the CRISPR/Cas12f1 complex, it was found that the Stem 1 part, Stem 2 part, and Stem 5 (R:AR2) part of the Cas12f1 guide RNA do not interact with the Cas12f1 dimer, and this is called a disordered region.

Background - CRISPR/Cas system expression vector design

Vector design overview

In order to use the CRISPR/Cas system for gene editing, a vector having a sequence encoding each component of the CRISPR/Cas system is introduced into a cell, and each component of the CRISPR/Cas system is expressed in the cell. It is widely used. Hereinafter, the components of the vector that allow the CRISPR/Cas system to be expressed in cells will be described.

Nucleic Acids Encoding CRISPR/Cas System Components

Since the purpose of the vector is to allow expression of each component of the CRISPR/Cas system in a cell, the sequence of the vector must necessarily include at least one of the nucleic acid sequences encoding each component of the CRISPR/Cas system. Specifically, the sequence of the vector includes a guide RNA included in the CRISPR/Cas system to be expressed, and/or a nucleic acid sequence encoding a Cas protein. In this case, the sequence of the vector encodes a nucleic acid sequence encoding a Cas12f1 guide RNA engineered according to the purpose and a codon-optimized Cas protein or an engineered Cas protein as well as a nucleic acid sequence encoding a wild-type guide RNA and a wild-type Cas protein. It may contain a nucleic acid sequence that

Regulating/Control Components

In order to express the vector in a cell, it must contain one or more regulatory/control elements. Specifically, the regulatory/control elements include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an Internal Ribosome Entry Site (IRES), a splice acceptor, a 2A sequence and / or may include a replication origin, but is not limited thereto. In this case, the origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and/or a BBV origin of replication, but is not limited thereto.

promoter

In order to express the expression target of the vector in a cell, a promoter sequence must be operatively linked to a sequence encoding each component so that the RNA transcription factor can be activated in the cell. The promoter sequence may be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can properly express the components of the CRISPR/Cas system in cells. The promoter sequence may be a promoter that promotes transcription of RNA polymerase (eg, RNA Pol I, Pol II, or Pol III). For example, the promoter is SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, such as CMV immediate early promoter region (CMVIE) cytomegalovirus (CMV) promoter, rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), enhanced U6 promoter (e.g., Xia et al. , Nucleic Acids Res. 2003 Sep 1;31(17)), human H1 promoter (H1), and 7SK, but is not limited thereto.

end signal

When the vector sequence includes the promoter sequence, transcription of a sequence operably linked to the promoter is induced by an RNA transcription factor. A sequence inducing transcription termination of the RNA transcription factor is referred to as a termination signal. The termination signal may vary depending on the type of promoter sequence. For example, when the promoter is a U6 or H1 promoter, the promoter recognizes a thymidine continuation sequence (eg, a TTTTTT (T6) sequence) as a termination signal.

additional expression elements

The vector may contain a nucleic acid sequence encoding an additional expression element to be expressed by a person skilled in the art other than the wild-type CRISPR/Cas system configuration and/or the engineered CRISPR/Cas system as needed. For example, the additional expression element may be, but is not limited to, one of the tags described in the “tag” section of “description of terms”. For example, the additional expression element is a herbicide resistance gene such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, It may be an antibiotic resistance gene such as G418, bleomycin, hygromycin, or chloramphenicol, but is not limited thereto.

Form of expression vector

The expression vector may be designed in the form of a linear or circular vector.

Limitations of the prior art

Since the CRISPR/Cas12f1 system is relatively small in size, it has been reported as an attractive system for gene editing technology (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018). )) is being studied (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018), US 2020/0190494 A1). However, gene editing activity in cells (eg, eukaryotic cells) does not appear or is too low, which is an obstacle to its utilization.

As a continuing study, in the recent prior literature (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)), Cas12f1 protein is a dimer in the CRISPR / Cas12f1 system. , and the structure of guide RNAs. It was found by the above literature that there is a so-called disordered region that does not directly interact with the Cas12f1 protein in the guide RNA portion, and another prior literature (Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, In bioRxiv (2020)), the disordered region was removed and double-stranded DNA cleavage efficiency was examined in vitro. However, the preceding studies do not 1) show or increase intracellular gene editing activity (eg, indel generation efficiency), and 2) there are experiments that remove the disordered region, but rather the cleavage activity is low. 3) Moreover, as the above experiment was conducted in vitro, it failed to reveal what modifications were made to show intracellular gene editing activity or to increase efficiency.

Therefore, it remains an important task to enhance the intracellular gene editing activity of the CRISPR/Cas12f1 system.

Characterization of engineered Cas12f1 guide RNAs

Engineered Cas12f1 Guide RNA Overview

The present specification provides an engineered Cas12f1 guide RNA for increasing the intracellular gene editing activity of the CRISPR/Cas12f1 system by overcoming the limitations of the prior art. The engineered Cas12f1 guide RNA is a modified guide RNA structure found in nature. The engineered Cas12f1 guide RNA is characterized in that at least a part of the scaffold region that interacts with the Cas12f1 protein is modified.

In one embodiment, the engineered Cas12f1 guide RNA may include an engineered scaffold region and a spacer. At this time, the engineered scaffold region is characterized in that it is different from the scaffold region of guide RNA found in nature.

Characteristics of Engineered Cas12f1 Guide RNAs - Modified at least one site in the scaffold region

The engineered Cas12f1 guide RNA provided herein is characterized in that a part of its scaffold portion is modified compared to the guide RNA found in nature. The scaffold region is a region containing tracrRNA and a portion of crRNA, and functions to interact with the Cas12f1 protein. The scaffold region will be described in more detail below.

In one embodiment, the engineered Cas12f1 guide RNA may include an engineered scaffold region. In this case, the engineered scaffold region is characterized in that the scaffold region of the guide RNA found in nature is modified. Therefore, the engineered scaffold region has a sequence different from that of the guide RNA found in nature. In one embodiment, the engineered scaffold region may be one in which some regions of the scaffold region of guide RNA found in nature have been removed. In one embodiment, the engineered scaffold region may have one or more nucleotides removed from the scaffold region of guide RNA found in nature.

Effect of engineered Cas12f1 guide RNA

When the engineered Cas12f1 guide RNA provided herein is used in the CRISPR/Cas12f1 system, the gene editing activity in cells is dramatically improved compared to when the guide RNA found in nature is used. The present inventors have revealed in detail what composition should be added to the guide RNA found in nature, or what kind of modification should be applied to the scaffold region to improve gene editing efficiency through experiments. By using the engineered Cas12f1 guide RNA, it is possible to edit genes with high efficiency in cells by overcoming the limitations of the prior art. In addition, the engineered Cas12f1 guide RNA has a length equal to or shorter than that of a guide RNA found in nature, so it has high application potential in the field of gene editing technology. If the engineered Cas12f1 guide RNA is used, the advantages of the CRISPR/Cas12f1 system (eg, the advantage of very small size) can be fully utilized in gene editing technology.

Uses of engineered Cas12f1 guide RNAs

The engineered Cas12f1 guide RNA provided herein can be used for gene editing and/or gene therapy together with Cas12f1 protein. In addition, the engineered Cas12f1 guide RNA can be used for preparing a composition for gene editing.

Explanation of Terms - Terms referring to the portion of the Cas12f1 guide RNA

Part of the Cas12f1 guide RNA - an overview

It is well known to those skilled in the art that Cas12f1 guide RNA found in nature is divided into tracrRNA and crRNA, and the crRNA can be further divided into a crRNA repeat sequence portion and a spacer.

Apart from the above criteria, in the present specification, the portion of the Cas12f1 guide RNA that interacts with the Cas12f1 protein is collectively referred to as a scaffold region. The scaffold region includes tracrRNA and a portion of crRNA, but does not necessarily refer to one molecule of RNA. The scaffold region may be subdivided into a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region. When the subdivided region is described on tracrRNA or crRNA, the first to fourth regions are included in tracrRNA, and the fifth to sixth regions are included in crRNA, specifically crRNA repeat sequence.

The "n-th region" or "n-th region found in nature" (n is an integer greater than or equal to 1 and less than or equal to 6) described below basically refers to each part of the Cas12f1 guide RNA found in nature. The region corresponding to the above classification criteria in the engineered Cas12f1 guide RNA is generally described as “modified n-th region” to “n-th region of engineered scaffold region”.

However, even in the n-th region included in the engineered scaffold region, there may be cases where it is not otherwise deformed and is the same as the n-th region found in nature. Only in that case, the term “n-th region” may be used interchangeably. In this case, the target referred to by the "n-th region" (eg, whether it is a region included in an engineered Cas12f1 guide RNA or a region included in a guide RNA found in nature) should be appropriately interpreted according to the context.

tracrRNA, crRNA

When used as "tracrRNA" or "crRNA" in the present specification, it includes all meanings that can be recognized by those skilled in the art of CRISPR/Cas. This is generally used as a term to refer to each molecule of dual guide RNA found in nature, but it can also be used to refer to each corresponding portion of a single guide RNA in which the tracrRNA and crRNA are linked by a linker. Unless otherwise specified, when only tracrRNA and crRNA are described, it means tracrRNA and crRNA constituting the CRISPR/Cas12f1 system.

일 구현예로, tracrRNA의 서열은 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (서열번호 1) 또는 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3' (서열번호 2)일 수 있다. In one embodiment, the tracrRNA comprises a first region, a second region, a third region, and a fourth region. In one embodiment, the tracrRNA is one in which the first region, the second region, the third region, and the fourth region are sequentially linked from the 5' end to the 3' end.

In one embodiment, the sequence of crRNA comprises a crRNA repeat sequence and a spacer sequence. In this case, the crRNA repeat sequence may be 5'-GAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 3) or 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 4). The crRNA repeat sequence comprises a fifth region and a sixth region. The spacer sequence may vary depending on the target sequence and generally comprises 10 to 50 nucleotides. In one embodiment, the crRNA is one in which a fifth region, a sixth region, and a spacer are sequentially linked in a direction from the 5' end to the 3' end.

Scaffold Area - Overview

When used herein as a “scaffold region”, it refers to the rest of the guide RNAs found in nature except for the spacer. Specifically, the scaffold region includes tracrRNA, and a portion of crRNA. Specifically, a portion of the crRNA may be a portion of a crRNA repeat sequence. The scaffold region is generally known as a portion capable of interacting with a Cas protein. In the present specification, the scaffold region is divided into first to sixth regions, and each region will be described in more detail below.

Scaffold region 1 - first region

When used herein as a "first region", it refers to a region including the 5' end of tracrRNA. The first region may include nucleotides forming a Stem structure in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto.

The first region includes a Stem 1 moiety (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The first region may include one or more nucleotides adjacent to the Stem 1 portion.

The first region comprises a disordered region in the CRISPR/Cas12f1 complex that does not interact with the Cas12f1 protein.

In one embodiment, the first region may mean from the 1st nucleotide to the 11th nucleotide from the 5' end of the tracrRNA represented by SEQ ID NO: 1 or 2. In one embodiment, the sequence of the first region may be 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9).

Scaffold region 2 - second region

When used herein as a "second region", it refers to a region located in the 3'-end direction of the first region in tracrRNA. The second region may include nucleotides forming a Stem structure in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto. In this case, the stem structure is different from the stem included in the first region.

The second region includes a Stem 2 moiety (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The second region may include one or more nucleotides adjacent to the Stem 2 portion.

The second region may include one or more nucleotides that interact with the RuvC domain of one Cas12f1 protein forming a dimer and/or the RuvC domain of another Cas12f1 protein forming a dimer in the CRISPR/Cas12f1 complex. The second region comprises a disordered region in the CRISPR/Cas12f1 complex that does not interact with the Cas12f1 protein.

In one embodiment, the second region may mean from the 22nd nucleotide to the 71st nucleotide from the 5' end of the tracrRNA represented by SEQ ID NO: 1 or 2. In one embodiment, the sequence of the second region may be 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 10).

Scaffold Region 3 - Third Region

When used herein as a "third region", it refers to a region located in the 3'-end direction of the second region in tracrRNA. The third region may include nucleotides forming a stem structure in the CRISPR/Cas12f1 complex and nucleotides forming a complementary bond with some nucleotides included in crRNA, and may include nucleotides adjacent thereto.

The third region consists of a Stem 4 portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) and a Stem 3-PK (R:AR-1) portion and nucleotides belonging to the heavy tracrRNA (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The third region may include one or more nucleotides adjacent to nucleotides belonging to tracrRNA among the Stem 4 portion and/or the Stem 3-PK (R:AR-1) portion.

The third region comprises one or more nucleotides that interact with the WED domain and/or the RuvC domain of one Cas12f1 protein forming a dimer in the CRISPR/Cas12f1 complex. In this case, the nucleotide may be a nucleotide belonging to tracrRNA in the Stem 3-PK (R:AR-1) portion.

The third region includes one or more nucleotides that interact with the RuvC domain of one Cas12f1 protein forming a dimer and/or the REC domain of another Cas12f1 protein forming a dimer in the CRISPR/Cas12f1 complex. In this case, the nucleotide may be a nucleotide included in the Stem 4 portion.

The third region comprises one or more nucleotides complementary to one or more nucleotides included in the sixth region of the crRNA.

In one embodiment, the third region may mean from the 72nd nucleotide to the 127th nucleotide from the 5' end of the tracrRNA represented by SEQ ID NO: 1 or 2. In one embodiment, the sequence of the third region may be 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11).

Scaffold Region 4 - Fourth Region

When used herein as a "fourth region", it refers to a region located at the 3' end of the third region of tracrRNA. The fourth region may include nucleotides capable of forming a complementary bond with some nucleotides included in crRNA in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto.

The fourth region includes a nucleotide belonging to tracrRNA in Stem 5 (R:AR-2) (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) ). The fourth region may include one or more nucleotides adjacent to a nucleotide belonging to tracrRNA in Stem 5 (R:AR-2).

The fourth region may include one or more nucleotides that interact with the WED domain and/or the ZF domain of one Cas12f1 protein forming a dimer in the CRISPR/Cas12f1 complex. In this case, the nucleotide may be a nucleotide belonging to tracrRNA in Stem 5 (R:AR-2).

The fourth region may include one or more nucleotides complementary to one or more nucleotides included in the fifth region of the crRNA. The fourth region comprises a disordered region in the CRISPR/Cas12f1 complex that does not interact with the Cas12f1 protein.

In one embodiment, the fourth region may mean from the 128th nucleotide to the 140th nucleotide from the 5' end of the tracrRNA represented by SEQ ID NO: 1. In one embodiment, the fourth region may mean from the 128th nucleotide to the 162th nucleotide from the 5' end of the tracrRNA represented by SEQ ID NO: 2.

In one embodiment, the sequence of the fourth region may be 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 12) or 5'-AACAAAUUCAUUUUUCCUCUCCCAAUUCUGCACAA-3' (SEQ ID NO: 13).

Scaffold Region 5 - Fifth Region

When used herein as a "fifth region", it refers to a region including the crRNA 5' end. The fifth region includes nucleotides that form a complementary bond with one or more nucleotides of the fourth region in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto.

The fifth region includes a nucleotide belonging to the crRNA of the Stem 5 (R:AR-2) portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021) )). The fifth region may include one or more nucleotides adjacent to a nucleotide belonging to the crRNA of the Stem 5 (R:AR-2) portion.

The fifth region may include one or more nucleotides that interact with the WED domain, the REC domain and/or the ZF domain of one Cas12f1 protein constituting a dimer in the CRISPR/Cas12f1 complex. In this case, the nucleotide may be a nucleotide belonging to crRNA in Stem 5 (R:AR-2).

The fifth region may include one or more nucleotides complementary to one or more nucleotides included in the fourth region. The fifth region comprises a disordered region in the CRISPR/Cas12f1 complex that does not interact with the Cas12f1 protein.

In one embodiment, the fifth region may mean from the 1st nucleotide to the 10th nucleotide from the 5' end of the crRNA represented by SEQ ID NO: 3. In one embodiment, the fifth region may mean from the 1st nucleotide to the 30th nucleotide from the 5' end of the crRNA represented by SEQ ID NO: 4. In one embodiment, the sequence of the fifth region may be 5'-GAAUGAAGGA-3' (SEQ ID NO: 14) or 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 15).

Scaffold Region 6 - Sixth Region

When used herein as a "sixth region", it refers to a region located in the 3'-end direction of the fifth region in crRNA. The sixth region includes nucleotides that form a complementary bond with one or more nucleotides of the third region in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto.

The sixth region includes a nucleotide belonging to crRNA in the PK (R:AR-1) portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) ). The sixth region may include one or more nucleotides adjacent to a nucleotide belonging to the crRNA of the PK (R:AR-1) portion.

The sixth region includes one or more nucleotides that interact with the WED domain, ZF domain and/or RuvC domain of one Cas12f1 protein constituting a dimer in the CRISPR/Cas12f1 complex. In this case, the nucleotide may be a nucleotide belonging to crRNA in the Stem 3-PK (R:AR-1) portion.

In one embodiment, the sixth region may mean from the 11th nucleotide to the 17th nucleotide from the 5' end of the crRNA represented by SEQ ID NO: 3. In one embodiment, the sixth region may mean from the 31st nucleotide to the 37th nucleotide from the 5' end of the crRNA represented by SEQ ID NO: 4. In one embodiment, the sequence of the sixth region may be 5'-AUGCAAC-3'.

spacer

When used herein, "spacer" refers to one or more nucleotides that hybridize with a portion of a target sequence in the CRISPR/Cas12f1 system. The spacer refers to 10 to 50 consecutive nucleotides near the 3' end of the crRNA of the guide RNA in the CRISPR/Cas12f1 system. The spacer is designed to correspond to the target sequence of the target nucleic acid to be edited using the CRISPR/Cas12f1 system. In other words, the spacer may have various sequences depending on the target sequence of the target nucleic acid.

Engineered Scaffold Area - Overview

Engineered Scaffold Area Overview

Herein, an engineered scaffold region that can be introduced to improve the gene editing efficiency of the CRISPR/Cas12f1 system is provided. The engineered scaffold region improves the gene editing efficiency of the CRISPR/Cas12f1 system in which the engineered Cas12f1 guide RNA is used. The engineered scaffold region is characterized in that it has a sequence and/or structure different from that of the scaffold region of Cas12f1 guide RNA found in nature (hereinafter, the scaffold region found in nature) is modified in one or more places. .

In this case, the function of the engineered scaffold region is the same as or has a function similar to that of the scaffold region found in nature. Specifically, the engineered scaffold region has the function of interacting with the Cas12f1 protein dimer of the CRISPR/Cas12f1 complex.

The engineered scaffold region includes regions corresponding to each portion of the scaffold region found in nature. Specifically, the engineered scaffold region includes a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region, which is the first region contained in the scaffold region found in nature. Each of the first to sixth areas corresponds to each other.

Modifications to make single guide RNAs

The engineered Cas12f1 guide RNA provided herein may be a single guide RNA molecule. Accordingly, the engineered scaffold region provided herein may be one in which one or more of each region is modified, and additionally, the 3' end of the tracrRNA fourth region and the 5' end of the crRNA fifth region may be linked through a linker.

In one embodiment, the engineered scaffold region is one or more modified in a scaffold region found in nature, and the 3' end of the fourth region and the 5' end of the fifth region are linked through a linker. can In this case, the linker may be 5'-GAAA-3'.

Engineered Scaffold Area 1 - First Area Variant

Overview of the first region transformation

The engineered scaffold region provided herein may be a first region modified from a scaffold region found in nature.

In one embodiment, the engineered scaffold region may include a deformed first region. In this case, the modified first region has one or more nucleotides removed from the first region of the scaffold region found in nature. In this case, the removed nucleotide is a nucleotide selected from the region forming the Stem structure in the CRISPR/Cas12f1 complex.

In one embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may include one in which one or more nucleotides included in the first region among scaffold regions found in nature have been removed. In one embodiment, the removed nucleotide may be a nucleotide included in a portion forming a stem structure in the CRISPR/Cas12f1 complex among the first regions found in nature. In one embodiment, the removed nucleotide is Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the first region found in nature. It may be a nucleotide belonging to In one embodiment, the removed nucleotide may be a nucleotide that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex among the first regions found in nature.

In one embodiment, the modified first region is characterized in that it comprises a 5'-A-3' sequence in the 3'-terminal direction.

In one embodiment, the engineered scaffold region may be one in which a first region among scaffold regions found in nature has been removed. In other words, the engineered scaffold region may not include a region corresponding to the first region among scaffold regions found in nature.

First region modification - some nucleotides removed

The first region of the engineered scaffold region may have one or more nucleotides removed from the first region of the scaffold region found in nature.

In one embodiment, the modified first region of the engineered scaffold region may have 1 to 20 nucleotides removed from the 5' end of the first region of the scaffold region found in nature. In one embodiment, the modified first region is 1, 2, 3, 4, 5, 6, 7, 8 at the 5' end of the first region of the scaffold region found in nature. 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides may be removed. In one embodiment, the modified first region may be one in which consecutive nucleotides within two numerical ranges selected in the previous sentence are removed from the 5' end of the first region of the scaffold region found in nature. For example, 1 to 3 consecutive nucleotides at the 5' end may be removed from the first region of the scaffold region found in nature.

In one embodiment, the modified first region comprises at least one nucleotide, which may be 5'-A-3'.

Modified first region sequence example

In one embodiment, the sequence of the modified first region is 5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA -3', 5'-GGAGAA-3', 5'-UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5 '-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20) , 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23) 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25), and 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26).

In one embodiment, the sequence of the engineered scaffold region in which the first region is modified may comprise:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 10,

SEQ ID NO: 11, and

sequence to which SEQ ID NO: 12 is linked; and

A sequence in which SEQ ID NO: 14 and 5'-AUGCAAC-3' are linked in the direction from the 5' end to the 3' end.

In one embodiment, the sequence of the engineered scaffold region in which the first region is modified may comprise:

5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 118), 5'-AACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 119), 5'-GAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 120), 5'-AGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 121 ), 5'-GAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 122), 5'-GGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 123), 5'-UGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 124), 5'-GUGGAGAACC GCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 125), 5'-AGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 126), 5'-AAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 127), 5'-AAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 128), 5 '-UAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 129), 5'-AUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 130), 5'-GAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAA AUUCAUUU-3'(서열번호 131), 5'-UGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 132), 5'-CUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 133), 5'-ACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 134), 5 '-CACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 135), 5'-UCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 136), 및 5'-UUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 137)로 이뤄진 군에서 선택된 서열; and 5'-GAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 3).

In one embodiment, the sequence of the engineered scaffold region in which the first region is modified may comprise:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', and a sequence selected from the group consisting of SEQ ID NOs: 16-26;

SEQ ID NO: 10,

SEQ ID NO: 11, and

sequence to which SEQ ID NO: 13 is linked; and

A sequence in which SEQ ID NOs: 15 and 5'-AUGCAAC-3' are linked in a direction from the 5' end to the 3' end.

In one embodiment, the sequence of the engineered scaffold region in which the first region is modified is in the 5' to 3' terminal direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', and a sequence selected from the group consisting of SEQ ID NOs: 16-26;

SEQ ID NO: 10,

SEQ ID NO: 11,

SEQ ID NO: 12,

linker,

SEQ ID NO: 14, and

5'-AUGCAAC-3' may be linked.

In this case, the linker may be 5'-GAAA-3'.

일 구현예로, 제1 영역이 변형된 엔지니어링 된 스캐폴드 영역의 서열은 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 167), 5'-AACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 168), 5'-GAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC- 3'(서열번호 169), 5'-AGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 170), 5'-GAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 171), 5'-GGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열 번호 172), 5'-UGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 173), 5'-GUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 174), 5'-AGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 175), 5'-AAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (서열번호 176), 5'-AAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 177), 5'-UAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC- 3'(서열번호 178), 5'-AUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 179), 5'-GAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 180), 5'-UGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 181), 5'- CUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 182), 5'-ACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 183), 5'-CACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAAC CCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 184), 5'-UCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 185), 및 5'-UUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 186)에서 선택된 서열일 수 있다.

Engineered Scaffold Region 2 - Second Region Variant

Overview of the second area transformation

The engineered scaffold region included in the engineered guide RNA provided herein may be a modified second region from the scaffold region found in nature.

In one embodiment, the engineered scaffold region may include a modified second region. In this case, the modified second region has one or more nucleotides removed from the second region of the scaffold region found in nature. In this case, the removed nucleotide is a nucleotide selected from the region forming the Stem structure in the CRISPR/Cas12f1 complex.

In one embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may include one in which one or more nucleotides included in the second region among scaffold regions found in nature have been removed. In one embodiment, the removal of the nucleotide may occur in a portion forming a stem structure among the second region found in the natural world, and the nucleotide may be removed in units of a base pair. In one embodiment, the removed nucleotide may be a nucleotide included in a portion forming a stem structure in the CRISPR/Cas12f1 complex among the second region found in nature. In one embodiment, the removed nucleotide is Stem 2 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the second region found in nature. It may be a nucleotide belonging to In one embodiment, the removed nucleotide may be a nucleotide that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex among the second region found in nature.

In one embodiment, the modified second region is characterized in that it has a 5'-CCGCUUCAC-3' (SEQ ID NO: 432) sequence in the 5'-terminal direction. In one embodiment, the modified second region is characterized in that it has a 5'-UGAGUGAAGG-3' (SEQ ID NO: 433) sequence in the 3' terminal direction.

Second Region Modification 1 - Remove some nucleotides

The second region of the engineered scaffold region may have one or more nucleotides removed from the second region of the scaffold region found in nature.

In one embodiment, the modified second region of the engineered scaffold region may have 1 to 27 nucleotides removed from the second region of the scaffold region found in nature. In one embodiment, the modified second region is, in the second region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 10, the 12th to 24th nucleotides from the 5' end, and/or the 27th to 40th One or more of the second nucleotides may be removed. In one embodiment, the modified second region is 1, 2, 3 of the 12th to 24th nucleotides from the 5' end based on the sequence of SEQ ID NO: 10 in the second region of the scaffold region found in nature. 5, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive nucleotides may be removed. In one embodiment, the modified second region is 1, 2, 3 of the 27th to 38th nucleotides from the 5' end, based on the sequence of SEQ ID NO: 10, in the second region of the scaffold region found in nature. Dogs, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive nucleotides may be removed.

In one embodiment, the sequence of the modified second region is characterized in that it comprises at least 5'-CCGCUUCAC-3' (SEQ ID NO: 432) and 5'-UGAGUGAAGG-3' (SEQ ID NO: 433). At this time, in the sequence of the modified second region, 5'-CCGCUUCAC-3' (SEQ ID NO: 432) and 5'-UGAGUGAAGG-3' (SEQ ID NO: 433) are linked in order from the 5' end to the 3' end. There may be, and may be linked through an appropriate intermediate sequence. In one example, the intermediate sequence is 5'-UUAG-3', 5'-AUUAGU-3', 5'-AAUUAGCU-3', 5'-AAAUUAGACU-3' (SEQ ID NO: 57), 5'-AAAGUUAGAACU-3 '(SEQ ID NO: 58), 5'-AAAGCUUAGGAACU-3' (SEQ ID NO: 59), 5'-AAAGCUUUAGAGAACU-3' (SEQ ID NO: 60), 5'-AAAGCUGUUAGUUAGAACU-3' (SEQ ID NO: 61), 5'-AAAGCUGUUAGUAGAACU -3' (SEQ ID NO: 62), 5'-AAAGCUGUUUAGAUUAGAACU-3' (SEQ ID NO: 63), 5'-AAAGCUGUCUUAGGAUUAGAACU-3' (SEQ ID NO: 64), 5'-AAAGCUGUGUCCUUAGGGAUUAGAACU-3' (SEQ ID NO: 65), 5' -AAAAGCUGUCCCUUAGGGGAUUAGAACUU-3' (SEQ ID NO: 434), and 5'-CAAAAGCUGUCCCUUAGGGGAUUAGAACUUG-3' (SEQ ID NO: 435) may be selected from the group consisting of.

2nd Area Variant 2 - Remove Base Pair Unit

The modification of the second region may be one in which one or more pairs of nucleotides complementary to each other included in the portion forming the stem structure are removed.

In one embodiment, the modified second region is the 12th to 24th nucleotides, and the 27th to 40th nucleotides from the 5' end, based on the sequence of SEQ ID NO: 10, in the second region of the scaffold region found in nature. In the CRISPR/Cas12f1 complex, one or more pairs of nucleotides constituting a base pair may be removed.

In one embodiment, the modified second region is the 12th to 24th nucleotides, and the 27th to 40th nucleotides from the 5' end, based on the sequence of SEQ ID NO: 10, in the second region of the scaffold region found in nature. In the CRISPR/Cas12f1 complex, one or more pairs of nucleotides forming a base pair and/or one or more nucleotides not forming a base pair may be removed.

In one embodiment, the modified second region is the 12th to 24th nucleotides, and the 27th to 40th nucleotides from the 5' end, based on the sequence of SEQ ID NO: 10, in the second region of the scaffold region found in nature. In the CRISPR/Cas12f1 complex, one or more pairs of nucleotides constituting a base pair and/or one or more pairs of mismatched nucleotides may be removed.

Modified second region sequence example

In one embodiment, the sequence of the modified second region is 5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUG-3' (SEQ ID NO: 431) 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGUG-3' (SEQ ID NO: 39), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUG-3' (SEQ ID NO: 41), 5'-CCGCUUCUGAGAAGG-3'-CCGCUUCUGAGAAGG-3' SEQ ID NO: 42), 5'-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 44), 5'-CCGCUUCUCACCAAAAGCUGUUAGUUAGAACUUGUAGUUAGUUAGAACUUGAGUGAAGGAAUGAGAG' (SEQ ID NO: 45), '(SEQ ID NO: 46), 5'-CCGCUUCACCAAAAGCUGUUUUAGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 48), and 5'-CCGCUUCACCAAUGAAUGUGUCUAGGGAA from group SEQ ID NO: 49) It may be a selected sequence.

In one embodiment, the sequence of the engineered scaffold region in which the second region is modified may comprise:

5' end to 3' end direction,

SEQ ID NO: 9,

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11, and

sequence to which SEQ ID NO: 12 is linked; and

A sequence in which SEQ ID NO: 14 and 5'-AUGCAAC-3' are linked in the direction from the 5' end to the 3' end.

In one embodiment, the sequence of the engineered scaffold region in which the second region is modified may comprise:

5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 138), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 139), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAUUAGACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 140), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 141 ), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 142), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 143), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 144), 5'- CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 145), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 146), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 147), 및 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3'(서열번호 148)로 a sequence selected from the group consisting of; and

SEQ ID NO: 3.

In one embodiment, the sequence of the engineered scaffold region in which the second region is modified may comprise:

5' end to 3' end direction,

SEQ ID NO: 9,

a sequence selected from SEQ ID NO: 38 to SEQ ID NO: 49, and SEQ ID NO: 430 to SEQ ID NO: 431;

SEQ ID NO: 11,

sequence to which SEQ ID NO: 13 is linked; and

A sequence in which SEQ ID NOs: 15 and 5'-AUGCAAC-3' are linked in a direction from the 5' end to the 3' end.

In one embodiment, the sequence of the engineered scaffold region in which the second region is modified is 5' to 3' end in SEQ ID NO: 9, SEQ ID NO: 38 to SEQ ID NO: 49, and SEQ ID NO: 430 to SEQ ID NO: 431 The selected sequence, SEQ ID NO: 11, SEQ ID NO: 12, linker, SEQ ID NO: 14, and 5'-AUGCAAC-3' may be linked.

In this case, the linker may be 5'-GAAA-3'.

일 구현예로, 제2 영역이 변형된 엔지니어링 된 스캐폴드 영역의 서열은 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 187), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 188), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAUUAGCUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC- 3'(서열번호 189), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 190), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 191), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 192), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 193), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 194), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 195), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'( 서열번호 196), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3'(서열번호 197), 및 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAU UCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 198) may be selected from the group consisting of.

Engineered Scaffold Region 3 - 3rd Region Variant

The third region in the engineered scaffold provided herein is a region containing a Stem 4 moiety that has a key effect on DNA cleavage activity. Accordingly, the third region of the engineered scaffold may be the same as the third region of the scaffold found in nature, or may be modified within the extent that the function of the third region is not impaired.

Engineered Scaffold Region 4 - 4th and 5th Region Variants

4th and 5th Region Variants Overview

The engineered scaffold region provided herein may be a modified region of the fourth and fifth regions from the scaffold region found in nature. Since the fourth region and the fifth region include a portion that hybridizes with each other in the CRISPR/Cas12f1 complex to constitute a stem, the corresponding portion may be modified together to constitute an engineered scaffold region.

In one embodiment, the engineered scaffold region may include a modified fourth region and/or a modified fifth region.

The modified fourth region is characterized in that one or more nucleotides have been removed from the fourth region of the scaffold region found in nature. The modified fifth region is characterized in that one or more nucleotides have been removed from the fifth region of the scaffold region found in nature.

In one embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may have one or more nucleotides removed from the fourth region and/or the fifth region among scaffold regions found in nature.

In one embodiment, the modified fourth region is characterized in that it has a 5'-AACAAA-3' sequence in the 5'-terminal direction. In one embodiment, the modified fifth region is characterized in that it has a 5'-GGA-3' sequence in the 3'-terminal direction.

4th and 5th Region Modifications 1 - Remove some nucleotides

The fourth region of the engineered scaffold region may be one in which one or more nucleotides have been removed from the fourth region among scaffold regions found in nature. The fifth region of the engineered scaffold region may have one or more nucleotides removed from the fifth region among scaffold regions found in nature.

In one embodiment, the modified fourth region of the engineered scaffold region may have 1 to 7 nucleotides removed from the fourth region of the scaffold region found in nature. In one embodiment, the modified fourth region of the engineered scaffold region may have 1 to 28 nucleotides removed from the fourth region of the scaffold region found in nature. In one embodiment, the modified fourth region may be one in which at least one of the 7th to 13th nucleotides from the 5' end has been removed from the fourth region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 12. have. In one embodiment, the modified fourth region may be one in which one or more of the 7th to 34th nucleotides from the 5' end have been removed from the fourth region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 13. have.

In one embodiment, the sequence of the modified fourth region is characterized in that it comprises at least 5'-AACAAA-3'.

In one embodiment, the modified fifth region of the engineered scaffold region may have 1 to 7 nucleotides removed from the fifth region of the scaffold region found in nature. In one embodiment, the modified fifth region of the engineered scaffold region may have 1 to 27 nucleotides removed from the fifth region of the scaffold region found in nature. In one embodiment, the modified fifth region may be one in which at least one of the 1st to 7th nucleotides from the 5' end has been removed from the fifth region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 14. have. In one embodiment, the modified fifth region may be one in which one or more of the 1st to 27th nucleotides from the 5' end have been removed from the fifth region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 15. have.

In one embodiment, the sequence of the modified fifth region comprises at least 5'-GGA-3'.

4th area and 5th area variation content 3 - Remove base pair unit

It is known that the fourth region and the fifth region complement each other in the CRISPR/Cas12 complex to form a stem. Since the above-described modification of the fourth region and the fifth region targets one or more nucleotides constituting the stem, the modification of the fourth region and the fifth region may be to remove the nucleotides constituting the stem in units of a base pair. have.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 12, the 7th to 13th nucleotides, and SEQ ID NO: 14 Based on the sequence, one or more pairs of nucleotides constituting a base pair in the CRISPR/Cas12f1 complex among the 1st to 7th nucleotides may be removed.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 12, the 7th to 13th nucleotides, and SEQ ID NO: 14 Based on the sequence, one or more pairs of nucleotides forming a base pair and/or one or more nucleotides not forming a base pair in the CRISPR/Cas12f1 complex among the 1st to 7th nucleotides may be removed.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 12, the 7th to 13th nucleotides, and SEQ ID NO: 14 Based on the sequence, one or more pairs of nucleotides constituting a base pair in the CRISPR/Cas12f1 complex among the first to seventh nucleotides and/or one or more pairs of mismatched nucleotides may be removed.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 13, the 7th to 34th nucleotides, and SEQ ID NO: Based on the 15th sequence, one or more pairs of nucleotides constituting a base pair in the CRISPR/Cas12f1 complex among the 1st to 27th nucleotides may be removed.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 13, the 7th to 34th nucleotides, and SEQ ID NO: Based on the 15th sequence, one or more pairs of nucleotides forming a base pair and/or one or more nucleotides not forming a base pair in the CRISPR/Cas12f1 complex among the 1st to 27th nucleotides may be removed.

In one embodiment, the modified fourth region and the fifth region are, in the fourth region and the fifth region of the scaffold region found in nature, based on SEQ ID NO: 13, the 7th to 34th nucleotides, and SEQ ID NO: Based on the 15th sequence, one or more pairs of nucleotides constituting a base pair in the CRISPR/Cas12f1 complex and/or one or more pairs of mismatched nucleotides among the 1st to 27th nucleotides may be removed.

Examples of Modified Fourth and Fifth Region Sequences

In one embodiment, the sequence of the modified fourth region is 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA It may be selected from the group consisting of -3' (SEQ ID NO: 66), 5'-AACAAAUUCAU-3' (SEQ ID NO: 67), and 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68).

In one embodiment, the sequence of the modified fourth region is 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA -3' (SEQ ID NO: 66), 5'-AACAAAUUCAU-3' (SEQ ID NO: 67), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 69), 5' -AACAAAUUCAUUUU-3' (SEQ ID NO: 70), 5'-AACAAAUUCAUUUUU-3' (SEQ ID NO: 71), 5'-AACAAAUUCAUUUUUC-3' (SEQ ID NO: 72), 5'-AACAAAUUCAUUUUUCC-3' (SEQ ID NO: 73), 5'-AACAAAUUCAUUUUUCCU-3' (SEQ ID NO: 74), 5'-AACAAAUUCAUUUUUCCUC-3' (SEQ ID NO: 75), 5'-AACAAAUUCAUUUUUUCCUCU-3' (SEQ ID NO: 76), 5'-AACAAAUUCAUUUUUCCUCUC-3' (SEQ ID NO: 77) ), 5'-AACAAAUUCAUUUUUCCUCUCC-3' (SEQ ID NO: 78), 5'-AACAAAUUCAUUUUUCCUCUCCA-3' (SEQ ID NO: 79), 5'-AACAAAUUCAUUUUUCCUCUCCAA-3' (SEQ ID NO: 80), 5'-AACAAAUUCAUUUUUCCUCUCUCCAAU-3' (SEQ ID NO: 80) No. 81), 5'-AACAAAUUCAUUUUUCCUCUCCAAUU-3' (SEQ ID NO: 82), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUC-3' (SEQ ID NO: 83), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUUCCUCUUCCAAUUCUAUUCUUCCAAUUCUAUUCUUCCUCUCUCUCU-3' (SEQ ID NO: 84), 5'-ACCAA (SEQ ID NO: 85), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGC-3' (SEQ ID NO: 86), 5'-AACAAAUUCAUUUUUCCUCUCCCAAUUCUGCA-3' (SEQ ID NO: 87), 5'-AAACAAAUUCAUUUUUCCUCUCUCCAUUCUCCCUCCAUAAUUG'-ACAUCAUCAUUCCUCCUCAAUUGAC-3' (SEQ ID NO: 86) It may be selected from the group consisting of A-3' (SEQ ID NO: 89).

In one embodiment, the sequence of the modified fifth region is 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA It may be selected from -3', 5'-AUGAAGGA-3', and 5'-AAUGAAGGA-3'.

In one embodiment, the sequence of the modified fifth region is 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA -3', 5'-AUGAAGGA-3', 5'-AAUGAAGGA-3', 5'-GAAUGAAGGA-3' (SEQ ID NO: 90), 5'-CGAAUGAAGGA-3' (SEQ ID NO: 91), 5'-ACGAAUGAAGGA -3' (SEQ ID NO: 92), 5'-GACGAAUGAAGGA-3' (SEQ ID NO: 93), 5'-AGACGAAUGAAGGA-3' (SEQ ID NO: 94), 5'-UAGACGAAUGAAGGA-3' (SEQ ID NO: 95), 5' -AUAGACGAAUGAAGGA-3' (SEQ ID NO: 96), 5'-AAUAGACGAAUGAAGGA-3' (SEQ ID NO: 97), 5'-GAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 98), 5'-CGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 99), 5'-CCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 100), 5'-CCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 101), 5'-ACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 102), 5'-AACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 103) ), 5'-GAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 104), 5'-AGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 105), 5'-CAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 106), 5'-GCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 105) 107), 5'-UGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 108), and 5'-UUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 109).

In one embodiment, the sequence of the engineered scaffold region in which the fourth region and the fifth region are modified may comprise:

5' end to 3' end direction,

SEQ ID NO: 9,

SEQ ID NO: 10,

SEQ ID NO: 11, and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', a sequence to which a sequence selected from the group consisting of SEQ ID NOs: 66 to 68 is linked; and

5' end to 3' end direction,

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', and 5 a sequence selected from the group consisting of '-AAUGAAGGA-3', and

5'-AUGCAAC-3' linked sequence.

In one embodiment, the sequence of the engineered scaffold region in which the fourth region and the fifth region are modified may comprise:

5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAA-3'(서열번호 149), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAU-3'(서열번호 150), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUU-3'(서열번호 151), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUC-3'(서열번호 152 ), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA-3'(서열번호 153), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAU-3'(서열번호 154), 및 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGC a sequence selected from the group consisting of UGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUU-3' (SEQ ID NO: 155); and

5'-GGAAUGCAAC-3', 5'-AGGAAUGCAAC-3', 5'-AAGGAAUGCAAC-3', 5'-GAAGGAAUGCAAC-3', 5'-UGAAGGAAUGCAAC-3', 5'-AUGAAGGAAUGCAAC-3', and 5 A sequence selected from the group consisting of '-AAUGAAGGAAUGCAAC-3'.

In one embodiment, the sequence of the engineered scaffold region in which the fourth region and the fifth region are modified may comprise:

5' end to 3' end direction,

SEQ ID NO: 9,

SEQ ID NO: 10,

SEQ ID NO: 11, and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', a sequence to which a sequence selected from the group consisting of SEQ ID NOs: 66 to 89 is linked; and

5' end to 3' end direction,

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', 5' -AAUGAAGGA-3', a sequence selected from the group consisting of SEQ ID NOs: 90-109, and

5'-AUGCAAC-3' linked sequence.

In one embodiment, the sequence of the engineered scaffold region in which the fourth region and the fifth region are modified is from the 5' end to the 3' end direction,

SEQ ID NO: 9,

SEQ ID NO: 10,

SEQ ID NO: 11, and

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), and 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and

5'-AUGCAAC-3' may be linked.

일 구현예로, 제4 영역 및 제5 영역이 변형된 엔지니어링 된 스캐폴드 영역의 서열은 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAAC-3'(서열번호 199), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUGAAAAGGAAUGCAAC-3'(서열번호 200), 5 '-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUGAAAAAGGAAUGCAAC-3'(서열번호 201), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCGAAAGAAGGAAUGCAAC-3'(서열번호 202), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGAAAUGAAGGAAUGCAAC-3'(서열번호 203), 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGC UUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUGAAAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 204), and 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGUGCAGUGGGCUGCUUGAAUCAGAUACCCUAAUGUCGAGAGAAGUGAAA number of columns selected from the group consisting of:

Engineered Scaffold Area 5 - 6th Area Variant

In the engineered scaffold provided herein, the sixth region is a region including nucleotides belonging to crRNA in the PK (R:AR-1) region. As described above, the sixth region includes one or more nucleotides that interact with the WED domain, the ZF domain and/or the RuvC domain of one Cas12f1 protein constituting a dimer in the CRISPR/Cas12f1 complex. The sixth region of the engineered scaffold may be the same as the sixth region of the scaffold found in nature, or may be modified within the extent that the function of the sixth region is not impaired.

Engineered Scaffold Area 6 - Combination of Each Variant

Combination overview of each variant

The engineered scaffold region included in the engineered Cas12f1 guide RNA provided herein may be a combination of one or more modifications for each region described above with a scaffold region found in nature.

In one embodiment, the engineered scaffold region may include a deformed first region and a deformed second region.

In one embodiment, the engineered scaffold region may include a deformed first region, and a deformed fourth region and a fifth region.

In one embodiment, the engineered scaffold region may include a modified second region, and a modified fourth region and a fifth region.

In one embodiment, the engineered scaffold region may include a deformed first region, a deformed second region, and a fourth and fifth deformed region.

In this case, the deformed region is the same as described in the section on deformation of each region.

Combination of each variant 1 - first region variant and second region variant

In one embodiment, the engineered scaffold region comprises a deformed first region and a deformed second region. In this case, the modified first region includes all the modifications described in the paragraph “Engineered scaffold region 1 - first region deformation”. In this case, the modified second region includes all of the modifications described in the paragraph “Engineered scaffold region 2 - second region deformation”.

In one embodiment, the engineered scaffold sequence in which the first region and the second region are modified may comprise:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11, and

sequence to which SEQ ID NO: 12 is linked; and

A sequence in which SEQ ID NO: 14 and 5'-AUGCAAC-3' are linked in the direction from the 5' end to the 3' end.

In one embodiment, the engineered scaffold sequence in which the first region and the second region are modified may comprise:

5' end to 3' end direction,

5'-ACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 156); and

5'-GAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 3).

In one embodiment, the sequence of the engineered scaffold region in which the first region and the second region are modified is in the 5' to 3' terminal direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11,

SEQ ID NO: 12,

linker,

SEQ ID NO: 14, and

5'-AUGCAAC-3' may be linked.

In this case, the linker may be 5'-GAAA-3'.

In one embodiment, the sequence of the engineered scaffold region in which the first region and the second region are modified may be 5'-ACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 206).

Combination of each variant 2 - first region variant and fourth region and fifth region variant

In one embodiment, the engineered scaffold region comprises a deformed first region, and a deformed fourth region and a fifth region. In this case, the modified first region includes all the modifications described in the paragraph “Engineered scaffold region 1 - first region deformation”. In this case, the modified fourth region and the fifth region include all of the modifications described in the paragraph “Engineered scaffold region 3 - fourth region and fifth region deformation”.

In one embodiment, the engineered scaffold sequence in which the first region and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 10,

SEQ ID NO: 11, and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', a sequence to which a sequence selected from the group consisting of SEQ ID NOs: 66 to 68 is linked; and

5' end to 3' end direction,

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', and 5 a sequence selected from the group consisting of '-AAUGAAGGA-3', and

5'-AUGCAAC-3' linked sequence.

In one embodiment, the engineered scaffold sequence in which the first region and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAA-3' (SEQ ID NO: 157); and

5'-GGAAUGCAAC-3' (SEQ ID NO: 160).

In one embodiment, the sequence of the engineered scaffold region in which the first region and the fourth region and the fifth region are modified is in the 5′ to 3′ terminal direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 10,

SEQ ID NO: 11,

a sequence selected from SEQ ID NOs: 110-116, and

5'-AUGCAAC-3' may be linked.

In one embodiment, the sequence of the engineered scaffold region in which the first region and the fourth region and the fifth region are modified may be 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAAC-3' (SEQ ID NO: 207).

Combination of each variant 3 - second region variant and fourth region and fifth region variant

In one embodiment, the engineered scaffold region comprises a modified second region and a modified fourth region and a fifth region. In this case, the modified second region includes all of the modifications described in the paragraph “Engineered scaffold region 2 - second region deformation”. In this case, the modified fourth region and the fifth region include all of the modifications described in the paragraph “Engineered scaffold region 3 - fourth region and fifth region deformation”.

In one embodiment, the engineered scaffold sequence in which the second region and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

SEQ ID NO: 9,

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11, and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', a sequence to which a sequence selected from the group consisting of SEQ ID NOs: 66 to 68 is linked; and

5' end to 3' end direction,

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', and 5 a sequence selected from the group consisting of '-AAUGAAGGA-3', and

5'-AUGCAAC-3' linked sequence.

In one embodiment, the engineered scaffold sequence in which the second region and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAA-3' (SEQ ID NO: 158); and

5'-GGAAUGCAAC-3' (SEQ ID NO: 160).

In one embodiment, the sequence of the engineered scaffold region in which the second region and the fourth region and the fifth region are modified is in the 5′ to 3′ terminal direction,

SEQ ID NO: 9,

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11,

a sequence selected from SEQ ID NOs: 110-116, and

5'-AUGCAAC-3' may be linked.

In one embodiment, the sequence of the engineered scaffold region in which the second and fourth and fifth regions are modified may be 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAAC-3' (SEQ ID NO:208).

Combination of each variant 4 - first region variant, second region variant, and fourth region and fifth region variant

In one embodiment, the engineered scaffold region comprises a deformed first region, a deformed second region, and a fourth and fifth deformed region. In this case, the modified first region includes all the modifications described in the paragraph “Engineered scaffold region 1 - first region deformation”. In this case, the modified second region includes all of the modifications described in the paragraph “Engineered scaffold region 2 - second region deformation”. In this case, the modified fourth region and the fifth region include all of the modifications described in the paragraph “Engineered scaffold region 3 - fourth region and fifth region deformation”.

In one embodiment, the engineered scaffold sequence in which the first region, the second region, and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11, and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', a sequence to which a sequence selected from the group consisting of SEQ ID NOs: 66 to 68 is linked; and

5' end to 3' end direction,

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', and 5 a sequence selected from the group consisting of '-AAUGAAGGA-3', and

5'-AUGCAAC-3' linked sequence.

In one embodiment, the engineered scaffold sequence in which the first region, the second region, and the fourth and fifth regions are modified may comprise:

5' end to 3' end direction,

5'-ACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAA-3' (SEQ ID NO: 159); and

5'-GGAAUGCAAC-3' (SEQ ID NO: 160).

In one embodiment, the sequence of the engineered scaffold region in which the first region, the second region and the fourth region and the fifth region are modified is in the 5′ to 3′ terminal direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', a sequence selected from the group consisting of SEQ ID NOs: 16 to 26;

SEQ ID NO: 38 to SEQ ID NO: 49, and a sequence selected from the group consisting of SEQ ID NO: 430 to SEQ ID NO: 431,

SEQ ID NO: 11,

a sequence selected from SEQ ID NOs: 110-116, and

5'-AUGCAAC-3' may be linked.

In one embodiment, the sequence of the engineered scaffold region in which the first region, the second region and the fourth region and the fifth region are modified may be 5'-ACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAAC-3' (SEQ ID NO: 209).

Combination of each variant 5 - the third region, and further variants of the sixth region

As described above, since the third region and the sixth region can also be deformed within a range in which their functions are not impaired, the engineered scaffold region provided herein is the first region, the second region, and the fourth region. , and/or in addition to the deformation of the fifth region, the third region and/or the sixth region may be additionally deformed.

Engineered Scaffold Region 7 - Contains Homologous Sequences

Engineered scaffold regions provided herein include "Engineered Scaffold Region 1 - First Region Variant", "Engineered Scaffold Region 2 - Second Region Variant", "Engineered Scaffold Region 3 - Third Region Variant" The engineered scaffold regions described in the paragraphs “Engineered Scaffold Region 4 - Fourth Region and Fifth Region Modifications”, and “Engineered Scaffold Region 5 - Combination of Each Variant” (hereinafter referred to as engineering scaffold regions). and a sequence homologous to the sequence of the scaffold region).

In one embodiment, the sequence of the engineered scaffold region is 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93% of any one of the sequences of the engineered scaffold region described above. , 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76 %, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% identical, or homologous sequences. In one embodiment, the scaffold sequence may be a sequence that matches any one of the sequences of the engineered scaffold region described above within two numerical ranges selected in the immediately preceding sentence. For example, the scaffold sequence may be a sequence that is 90% to 100% identical to any one of the sequences of the engineered scaffold region described above.

Engineered Cas12f1 guide RNA

Engineered Cas12f1 Guide RNA Overview

Herein, an engineered Cas12f1 guide RNA is provided to increase the intracellular gene editing efficiency of the CRISPR/Cas12f1 system. The engineered Cas12f1 guide RNA comprises an engineered scaffold, and a spacer. In this case, the engineered scaffold may be any one of those described in the above-mentioned "engineered scaffold region".

Single guide RNA or dual guide RNA

The engineered Cas12f1 guide RNA may be a single guide RNA or a dual guide RNA. The dual guide RNA means that the guide RNA is composed of two molecular RNAs, tracrRNA and crRNA. The single guide RNA means that the 3' end of the (engineered) tracrRNA and the 5' end of the (engineered) crRNA are connected through a linker. In other words, in the dual guide RNA, it means that the 3' end of the fourth region and the 5' end of the fifth region included in the engineered scaffold are linked through a linker. In this case, each region of the engineered scaffold may include all of the modifications described in the “Engineered scaffold region” paragraphs and a specific sequence thereof.

Engineered single guide RNA Example 1

In one embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region and a spacer are connected in order from the 5' end to the 3' end.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The engineered scaffold region is a first region, a second region, a third region, a fourth region, a linker, a fifth region, The six regions are sequentially connected, and one or more regions selected from the first region, the second region, the fourth region, and the fifth region may be modified compared to the scaffold region found in nature.

For example, when the first region of the engineered scaffold region is modified, the modified first region may have one or more nucleotides removed from the first region of the scaffold region found in nature. In this case, the removed nucleotide may be a nucleotide belonging to Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the first region. In this case, the sequence of the modified first region is characterized in that it includes 5'-A-3'.

As another example, when the second region of the engineered scaffold region is modified, the modified second region may have one or more nucleotides removed from the second region of the scaffold region found in nature. At this time, the removal of the nucleotide occurred in the portion forming the Stem 2 structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) of the second region. and nucleotides constituting a base pair with each other may be removed in pairs. In this case, the sequence of the modified second region is characterized in that it comprises at least 5'-CCGCUUCAC-3' (SEQ ID NO: 432) and 5'-UGAGUGAAGG-3' (SEQ ID NO: 433). More specifically, the sequence of the modified second region is 5'-CCGCUUCAC-3' (SEQ ID NO: 432) and 5'-UGAGUGAAGG-3' (SEQ ID NO: 433) in the order from the 5' end to the 3' end. They may be linked, and may be linked through an appropriate intermediate sequence. In one example, the intermediate sequence is 5'-UUAG-3', 5'-AUUAGU-3', 5'-AAUUAGCU-3', 5'-AAAUUAGACU-3' (SEQ ID NO: 57), 5'-AAAGUUAGAACU-3 '(SEQ ID NO: 58), 5'-AAAGCUUAGGAACU-3' (SEQ ID NO: 59), 5'-AAAGCUUUAGAGAACU-3' (SEQ ID NO: 60), 5'-AAAGCUGUUAGUUAGAACU-3' (SEQ ID NO: 61), 5'-AAAGCUGUUAGUAGAACU -3' (SEQ ID NO: 62), 5'-AAAGCUGUUUAGAUUAGAACU-3' (SEQ ID NO: 63), 5'-AAAGCUGUCUUAGGAUUAGAACU-3' (SEQ ID NO: 64), 5'-AAAGCUGUGUCCUUAGGGAUUAGAACU-3' (SEQ ID NO: 65), 5' -AAAAGCUGUCCCUUAGGGGAUUAGAACUU-3' (SEQ ID NO: 434), and 5'-CAAAAGCUGUCCCUUAGGGGAUUAGAACUUG-3' (SEQ ID NO: 435) may be selected from the group consisting of.

As another example, when the fourth region and the fifth region of the engineered scaffold region are modified, the modified fourth region and the fifth region are the fourth region and/or the fifth region of the scaffold region found in nature. One or more nucleotides of the region may be removed. At this time, the removal of the nucleotides is a Stem 5 (R:AR-2) structure of the fourth region and the fifth region (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1- 13 (2021)), and nucleotides constituting a base pair with each other may be removed in pairs. In this case, the sequence of the modified fourth region is characterized in that it includes at least 5'-AACAAA-3'. In this case, the sequence of the modified fifth region is characterized in that it includes at least 5'-GGA-3'.

Engineered Single Guide RNA Example 2

In one embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region and a spacer are connected in order from the 5' end to the 3' end.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region is one in which the following sequences are linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and

5'-AUGCAAC-3',

At this time, the sequence of the engineered scaffold region is different from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAACAAUGAAGGAA sequence number 7).

Engineered Single Guide RNA Example 3

In one embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region and a spacer are connected in order from the 5' end to the 3' end.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region is one in which the following sequences are linked in order from the 5' end to the 3' end:

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) No. 117), 5'-AACAAAUUCAUUUUGAAACGAAUGAAGGA-3' (SEQ ID NO: 293), 5'-AACAAAUUCAUUUUUGAAAACGAAUGAAGGA-3' (SEQ ID NO: 294), 5'-AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAAU-GAAAAAGGAUGA (SEQ ID NO: 296), 5'-AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 297), 5'-AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 298), 5'-AACAAAUUCAUUCAUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAACUGAAUGAAUUUUUCCUCUGAAUGAAGAAUUCA 3' (SEQ ID NO: 300), 5'-AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 301), 5'-AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAGU-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAU AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 304), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 305), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 306), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 307), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 308 ), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 309), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 310), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 311), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열 312), 5'-AACAAAUUCAUUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 313), and 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO:314) selected from the group consisting of; and

5'-AUGCAAC-3',

At this time, the sequence of the engineered scaffold region is different from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCUCGGAAAGUAACCCUCGAAACAAAUUCAUUCAUUGAACCUCGAAUCAUUGAACAAUUCCAACCCUCGAAACAAAUUGACAGUCAUCAUGACAUUGAACAAUUCA with the sequence that is different from the sequence.

Engineered Single Guide RNA Example 4

In one embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region and a spacer are connected in order from the 5' end to the 3' end.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region is one in which the following sequences are linked in order from the 5' end to the 3' end:

a first sequence represented by 5'-A-3';

a second sequence represented by 5'-CCGCUUCAC-3' (SEQ ID NO: 432);

a third sequence represented by 5'-UUAG-3';

a fourth sequence represented by 5'-UGAGUGAAGG-3' (SEQ ID NO: 433);

a fifth sequence represented by 5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

a sixth sequence represented by 5'-AACAAA-3';

linker;

a seventh sequence represented by 5'-GGA-3'; and

The eighth sequence represented by 5'-AUGCAAC-3'.

For example, the linker may be 5'-GAAA-3'.

In another example, the linker is 5'-GAAA-3', 5'-UGAAAA-3', 5'-UUGAAAAA-3', 5'-UUCGAAAGAA-3' (SEQ ID NO: 425), 5'-UUCAGAAAUGAA-3 In the group consisting of '(SEQ ID NO: 426), 5'-UUCAUGAAAAUGAA-3' (SEQ ID NO: 427), 5'-UUCAUUGAAAAAUGAA-3' (SEQ ID NO: 428), and 5'-UUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 429) may be selected.

In one embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-GA-3', 5'-AGA-3', 5'-GAGA-3', 5 '-GGAGA-3', 5'-UGGAGA-3', 5'-GUGGAGA-3', 5'-AGUGGAGA-3', 5'-AAUGGAGA-3', 5'-AAAGUGGAGA-3' (SEQ ID NO: 27 ), 5'-UAAAAGUGGAGA-3' (SEQ ID NO: 28), 5'-AUAAAGUGGAGA-3' (SEQ ID NO: 29), 5'-GAUAAAGUGGAGA-3' (SEQ ID NO: 30), 5'-UGAUAAAGUGGAGA-3' (SEQ ID NO: 30) No. 31), 5'-CUGAUAAAAGUGGAGA-3' (SEQ ID NO: 32), 5'-ACUGAUAAAGUGGAGA-3' (SEQ ID NO: 33), 5'-CACUGAUAAAAGUGGAGA-3' (SEQ ID NO: 34), 5'-UCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 35), 5'-UUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 36), and 5'-CUUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 37). It may further include a ninth sequence selected from the group consisting of. In this case, the 3' end of the ninth sequence may be linked to the 5' end of the first sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-AAA-3', 5'-AAAG-3', 5'-AAAGC-3', 5'-AAAGCU-3', 5'-AAAGCUG-3', 5'-AAAGCUGU-3', 5'-AAAGCUGUC-3', 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), and 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441) a tenth sequence selected from the group consisting of may additionally include. In this case, the 3' end of the second sequence and the 5' end of the third sequence may be connected through the 10th sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-CU-3', 5'-ACU-3', 5'-AACU-3', 5'-GAACU-3', 5'-AGAACU-3', 5'-UAGAACU-3', 5'-UUAGAACU-3', 5'-AUUAGAACU-3', 5'-GAUUAGAACU-3' (SEQ ID NO: 54), 5'-GGAUUAGAACU-3' (SEQ ID NO: 55), 5'-GGGAUUAGAACU-3' (SEQ ID NO: 56), 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442), and 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443) may additionally include an 11th sequence selected from the group consisting of. In this case, the 3' end of the third sequence and the 5' end of the fourth sequence may be connected through the eleventh sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-AAA-3', 5'-AAAG-3', 5'-AAAGC-3', 5'-AAAGCU-3', 5'-AAAGCUG-3', 5'-AAAGCUGU-3', 5'-AAAGCUGUC-3', 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), and 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441) a tenth sequence selected from the group consisting of and 5'-U-3', 5'-CU-3', 5'-ACU-3', 5'-AACU-3', 5'-GAACU-3', 5'-AGAACU-3', 5 '-UAGAACU-3', 5'-UUAGAACU-3', 5'-AUUAGAACU-3', 5'-GAUUAGAACU-3' (SEQ ID NO: 54), 5'-GGAUUAGAACU-3' (SEQ ID NO: 55), 5 '-GGGAUUAGAACU-3' (SEQ ID NO: 56), 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442), and 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443) further comprising an 11th sequence selected from the group consisting of can In this case, the 3' end of the second sequence and the 5' end of the third sequence are connected through the 10th sequence, and the 3' end of the third sequence and the 5' end of the fourth sequence are 11 may be linked through a sequence.

For example, when the tenth sequence is 5'-A-3', the eleventh sequence may be 5'-U-3'. As another example, when the tenth sequence is 5'-AA-3', the eleventh sequence may be 5'-CU-3'. As another example, when the tenth sequence is 5'-AAA-3', the eleventh sequence may be 5'-ACU-3'. As another example, when the tenth sequence is 5'-AAAG-3', the eleventh sequence may be 5'-AACU-3'. As another example, when the tenth sequence is 5'-AAAGC-3', the eleventh sequence may be 5'-GAACU-3'. As another example, when the tenth sequence is 5'-AAAGCU-3', the eleventh sequence may be 5'-AGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUG-3', the eleventh sequence may be 5'-UAGAACU-3' or 5'-UUAGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUGU-3', the eleventh sequence may be 5'-AUUAGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUGUC-3', the eleventh sequence may be 5'-GAUUAGAACU-3' (SEQ ID NO: 54). As another example, when the tenth sequence is 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), the eleventh sequence may be 5'-GGAUUAGAACU-3' (SEQ ID NO: 55). As another example, when the tenth sequence is 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), the eleventh sequence may be 5'-GGGAUUAGAACU-3' (SEQ ID NO: 56). As another example, when the tenth sequence is 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), the eleventh sequence may be 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442). As another example, when the tenth sequence is 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441), the eleventh sequence may be 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443).

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-UU-3', 5'-UUC-3', 5'-UUCA-3', It may additionally include a twelfth sequence selected from the group consisting of 5'-UUCAU-3', 5'-UUCAUU-3', and 5'-UUCAUUU-3'. In this case, the 3' end of the sixth sequence and the 5' end of the linker may be connected through the twelfth sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-UGAA-3', It may additionally include a 13th sequence selected from the group consisting of 5'-AUGAA-3', 5'-AAUGAA-3', and 5'-GAAUGAA-3'. In this case, the 3' end of the linker and the 5' end of the seventh sequence may be connected through the thirteenth sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-UU-3', 5'-UUC-3', 5'-UUCA-3', 12th sequence selected from the group consisting of 5'-UUCAU-3', 5'-UUCAUU-3', and 5'-UUCAUUU-3', and 5'-A-3', 5'-AA-3', A 13th sequence selected from the group consisting of 5'-GAA-3', 5'-UGAA-3', 5'-AUGAA-3', 5'-AAUGAA-3', and 5'-GAAUGAA-3' is additionally added may include In this case, the 3' end of the sixth sequence and the 5' end of the linker are connected through the twelfth sequence, and the 3' end of the linker and the 5' end of the seventh sequence are connected through the thirteenth sequence may have been

For example, when the twelfth sequence is 5'-U-3', the thirteenth sequence may be 5'-A-3'. As another example, when the twelfth sequence is 5'-UU-3', the thirteenth sequence may be 5'-AA-3'. As another example, when the twelfth sequence is 5'-UUC-3', the thirteenth sequence may be 5'-GAA-3'. As another example, when the twelfth sequence is 5'-UUCA-3', the thirteenth sequence may be 5'-UGAA-3'. As another example, when the twelfth sequence is 5'-UUCAU-3', the thirteenth sequence may be 5'-AUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUU-3', the thirteenth sequence may be 5'-AAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUU-3', the thirteenth sequence may be 5'-GAAUGAA-3'.

Example of engineered single guide RNA 5

In one embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region and a spacer are connected in order from the 5' end to the 3' end.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region is one in which the following sequences are linked in order from the 5' end to the 3' end:

a first sequence represented by 5'-A-3';

a second sequence represented by 5'-CCGCUUCAC-3' (SEQ ID NO: 432);

a third sequence represented by 5'-UUAG-3';

a fourth sequence represented by 5'-UGAGUGAAGG-3' (SEQ ID NO: 433);

a fifth sequence represented by 5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);

a sixth sequence represented by 5'-AACAAA-3';

linker;

a seventh sequence represented by 5'-GGA-3'; and

The eighth sequence represented by 5'-AUGCAAC-3'.

For example, the linker may be 5'-GAAA-3'.

In another example, the linker is 5'-GAAA-3', 5'-AGAAAG-3', 5'-AAGAAAGU-3', 5'-CAAGAAAGUU-3' (SEQ ID NO: 316), 5'-ACAAGAAAGUUG-3 '(SEQ ID NO: 317), 5'-CACAAGAAAGUUGC-3' (SEQ ID NO: 318), 5'-GCACAAGAAAGUUGCA-3' (SEQ ID NO: 319), 5'-UGCACAAGAAAGUUGCAG-3' (SEQ ID NO: 320), 5'-CUGCACAAGAAAGUUGCAGA -3' (SEQ ID NO: 321), 5'-UCUGCACAAGAAAGUUGCAGAA-3' (SEQ ID NO: 322), 5'-UUCUGCACAAAGAAAGUUGCAGAAC-3' (SEQ ID NO: 323), 5'-AUUCUGCACAAAGAAAGUUGCAGAACC-3' (SEQ ID NO: 324), 5' -AAUUCUGCACAAAGAAAGUUGCAGAACCC-3' (SEQ ID NO: 325), 5'-CAAUUCUGCACAAGAAAGUUGCAGAACCCG-3' (SEQ ID NO: 326), 5'-CCAAUUCUGCACAGAAAGUUGCAGAACCCGA-3' (SEQ ID NO: 327), 5'-UCCAAUUCUGCAGAACCCGAA (SEQ ID NO: 327), 5'-UCCAAUUCUGCAACCAAGAAA 5'-CUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAU-3' (SEQ ID NO: 329), 5'-UCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUA-3' (SEQ ID NO: 330), 5'-CUCUCCAAUUCUGCACAAUCGAAAGUUGCAGAACAAGAUAGUAG-3' (SEQ ID NO: 3CUGCAGAACAAGAAUGUAG-3' (SEQ ID NO: 331) ), 5'-UCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGAC-3' (SEQ ID NO: 333), 5'-UUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACG-3' (SEQ ID NO: 334), 5'-UUUCCUCUCCCAAUUCUGCACAAGAAAGUUGCA-3' (SEQ ID NO: 5), ACCCAGAAAGUUGCA -UUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAA-3'(서열번호 336), 5'-UUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAU-3'(서열번호 337), 5'-AUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUG-3'(서열번호 338), 5'-CAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGA-3'(서열번호 339), 5'-UCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 340), 5'-UCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAUGA-3' (SEQ ID NO: 341), and 5'-GACUAUUGAAUGA-3' (SEQ ID NO: 341).

In one embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-GA-3', 5'-AGA-3', 5'-GAGA-3', 5 '-GGAGA-3', 5'-UGGAGA-3', 5'-GUGGAGA-3', 5'-AGUGGAGA-3', 5'-AAUGGAGA-3', 5'-AAAGUGGAGA-3' (SEQ ID NO: 27 ), 5'-UAAAAGUGGAGA-3' (SEQ ID NO: 28), 5'-AUAAAGUGGAGA-3' (SEQ ID NO: 29), 5'-GAUAAAGUGGAGA-3' (SEQ ID NO: 30), 5'-UGAUAAAGUGGAGA-3' (SEQ ID NO: 30) No. 31), 5'-CUGAUAAAAGUGGAGA-3' (SEQ ID NO: 32), 5'-ACUGAUAAAGUGGAGA-3' (SEQ ID NO: 33), 5'-CACUGAUAAAAGUGGAGA-3' (SEQ ID NO: 34), 5'-UCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 35), 5'-UUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 36), and 5'-CUUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 37). It may further include a ninth sequence selected from the group consisting of. In this case, the 3' end of the ninth sequence may be linked to the 5' end of the first sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-AAA-3', 5'-AAAG-3', 5'-AAAGC-3', 5'-AAAGCU-3', 5'-AAAGCUG-3', 5'-AAAGCUGU-3', 5'-AAAGCUGUC-3', 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), and 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441) a tenth sequence selected from the group consisting of may additionally include. In this case, the 3' end of the second sequence and the 5' end of the third sequence may be connected through the 10th sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-CU-3', 5'-ACU-3', 5'-AACU-3', 5'-GAACU-3', 5'-AGAACU-3', 5'-UAGAACU-3', 5'-UUAGAACU-3', 5'-AUUAGAACU-3', 5'-GAUUAGAACU-3' (SEQ ID NO: 54), 5'-GGAUUAGAACU-3' (SEQ ID NO: 55), 5'-GGGAUUAGAACU-3' (SEQ ID NO: 56), 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442), and 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443) may additionally include an 11th sequence selected from the group consisting of. In this case, the 3' end of the third sequence and the 5' end of the fourth sequence may be connected through the eleventh sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-AAA-3', 5'-AAAG-3', 5'-AAAGC-3', 5'-AAAGCU-3', 5'-AAAGCUG-3', 5'-AAAGCUGU-3', 5'-AAAGCUGUC-3', 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), and 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441) a tenth sequence selected from the group consisting of and 5'-U-3', 5'-CU-3', 5'-ACU-3', 5'-AACU-3', 5'-GAACU-3', 5'-AGAACU-3', 5 '-UAGAACU-3', 5'-UUAGAACU-3', 5'-AUUAGAACU-3', 5'-GAUUAGAACU-3' (SEQ ID NO: 54), 5'-GGAUUAGAACU-3' (SEQ ID NO: 55), 5 '-GGGAUUAGAACU-3' (SEQ ID NO: 56), 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442), and 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443) further comprising an 11th sequence selected from the group consisting of can In this case, the 3' end of the second sequence and the 5' end of the third sequence are connected through the 10th sequence, and the 3' end of the third sequence and the 5' end of the fourth sequence are 11 may be linked through a sequence.

For example, when the tenth sequence is 5'-A-3', the eleventh sequence may be 5'-U-3'. As another example, when the tenth sequence is 5'-AA-3', the eleventh sequence may be 5'-CU-3'. As another example, when the tenth sequence is 5'-AAA-3', the eleventh sequence may be 5'-ACU-3'. As another example, when the tenth sequence is 5'-AAAG-3', the eleventh sequence may be 5'-AACU-3'. As another example, when the tenth sequence is 5'-AAAGC-3', the eleventh sequence may be 5'-GAACU-3'. As another example, when the tenth sequence is 5'-AAAGCU-3', the eleventh sequence may be 5'-AGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUG-3', the eleventh sequence may be 5'-UAGAACU-3' or 5'-UUAGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUGU-3', the eleventh sequence may be 5'-AUUAGAACU-3'. As another example, when the tenth sequence is 5'-AAAGCUGUC-3', the eleventh sequence may be 5'-GAUUAGAACU-3' (SEQ ID NO: 54). As another example, when the tenth sequence is 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), the eleventh sequence may be 5'-GGAUUAGAACU-3' (SEQ ID NO: 55). As another example, when the tenth sequence is 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), the eleventh sequence may be 5'-GGGAUUAGAACU-3' (SEQ ID NO: 56). As another example, when the tenth sequence is 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), the eleventh sequence may be 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442). As another example, when the tenth sequence is 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441), the eleventh sequence may be 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443).

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-UU-3', 5'-UUC-3', 5'-UUCA-3', 5'-UUCAU-3', 5'-UUCAUU-3', 5'-UUCAUUU-3', 5'-UUCAUUUU-3', 5'-UUCAUUUUU-3', 5'-UUCAUUUUUC-3' (SEQ ID NO: 343), 5'-UUCAUUUUUCC-3' (SEQ ID NO: 344), 5'-UUCAUUUUUCCU-3' (SEQ ID NO: 345), 5'-UUCAUUUUUCCUC-3' (SEQ ID NO: 346), 5'-UUCAUUUUUCCUCU-3' ( SEQ ID NO: 347), 5'-UUCAUUUUUCCUCUC-3' (SEQ ID NO: 348), 5'-UUCAUUUUUCCUCUCC-3' (SEQ ID NO: 349), 5'-UUCAUUUUUCCUCUCCA-3' (SEQ ID NO: 350), 5'-UUCAUUUUUCCUCUCCAA-3 '(SEQ ID NO: 351), 5'-UUCAUUUUUCCUCUCCAAU-3' (SEQ ID NO: 352), 5'-UUCAUUUUUCCUCUCCAAUU-3' (SEQ ID NO: 353), 5'-UUCAUUUUUCCUCUCCCAAUCUCU-3' (SEQ ID NO: 354), 5'-UUCCUCCAUUUUCCUCUCCAUUUU -3' (SEQ ID NO: 355), 5'-UUCAUUUUUCCUCCCAAUUCUG-3' (SEQ ID NO: 356), 5'-UUCAUUUUUCCUCCCAAUUCUGC-3' (SEQ ID NO: 357), 5'-UUCAUUUUUCCUCCUCCAAUUCUGCA-3' (SEQ ID NO: 358), 5' -UUCAUUUUUCCUCUCCCAAUUCUGCAC-3' (SEQ ID NO: 359), 5'-UUCAUUUUUCCUCUCCAAUUCUGCACA-3' (SEQ ID NO: 360), and 5'-UUCAUUUUUCCUCUCCCAAUUCUGCACAA-3' (SEQ ID NO: 361) may additionally include a twelfth sequence selected from the group consisting of have. In this case, the 3' end of the sixth sequence and the 5' end of the linker may be connected through the twelfth sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-UGAA-3', 5'-AUGAA-3', 5'-AAUGAA-3', 5'-GAAUGAA-3', 5'-CGAAUGAA-3', 5'-ACGAAUGAA-3', 5'-GACGAAUGAA-3' (SEQ ID NO: 362), 5'-AGACGAAUGAA-3' (SEQ ID NO: 363), 5'-UAGACGAAUGAA-3' (SEQ ID NO: 364), 5'-AUAGACGAAUGAA-3' (SEQ ID NO: 365), 5'-AAUAGACGAAUGAA-3' ( SEQ ID NO: 366), 5'-GAAUAGACGAAUGAA-3' (SEQ ID NO: 367), 5'-CGAAUAGACGAAUGAA-3' (SEQ ID NO: 368), 5'-CCGAAUAGACGAAUGAA-3' (SEQ ID NO: 369), 5'-CCCGAAUAGACGAAUGAA-3 '(SEQ ID NO: 370), 5'-ACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 371), 5'-AACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 372), 5'-GAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 373), 5'-AGAACCCGAAUAGACGAAUGAA -3' (SEQ ID NO: 374), 5'-CAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 375), 5'-GCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 376), 5'-UGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 377), 5' -UUGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 378), and 5'-GUUGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 379) may additionally include a thirteenth sequence selected from the group consisting of. In this case, the 3' end of the linker and the 5' end of the seventh sequence may be connected through the thirteenth sequence.

In another embodiment of the above embodiment, the sequence of the engineered scaffold region is 5'-U-3', 5'-UU-3', 5'-UUC-3', 5'-UUCA-3', 5'-UUCAU-3', 5'-UUCAUU-3', 5'-UUCAUUU-3', 5'-UUCAUUUU-3', 5'-UUCAUUUUU-3', 5'-UUCAUUUUUC-3' (SEQ ID NO: 343), 5'-UUCAUUUUUCC-3' (SEQ ID NO: 344), 5'-UUCAUUUUUCCU-3' (SEQ ID NO: 345), 5'-UUCAUUUUUCCUC-3' (SEQ ID NO: 346), 5'-UUCAUUUUUCCUCU-3' ( SEQ ID NO: 347), 5'-UUCAUUUUUCCUCUC-3' (SEQ ID NO: 348), 5'-UUCAUUUUUCCUCUCC-3' (SEQ ID NO: 349), 5'-UUCAUUUUUCCUCUCCA-3' (SEQ ID NO: 350), 5'-UUCAUUUUUCCUCUCCAA-3 '(SEQ ID NO: 351), 5'-UUCAUUUUUCCUCUCCAAU-3' (SEQ ID NO: 352), 5'-UUCAUUUUUCCUCUCCAAUU-3' (SEQ ID NO: 353), 5'-UUCAUUUUUCCUCUCCCAAUCUCU-3' (SEQ ID NO: 354), 5'-UUCCUCCAUUUUCCUCUCCAUUUU -3' (SEQ ID NO: 355), 5'-UUCAUUUUUCCUCCCAAUUCUG-3' (SEQ ID NO: 356), 5'-UUCAUUUUUCCUCCCAAUUCUGC-3' (SEQ ID NO: 357), 5'-UUCAUUUUUCCUCCUCCAAUUCUGCA-3' (SEQ ID NO: 358), 5' -UUCAUUUUUCCUCUCCCAAUUCUGCAC-3' (SEQ ID NO: 359), 5'-UUCAUUUUUCCUCUCCAAUUCUGCACA-3' (SEQ ID NO: 360), and 5'-UUCAUUUUUCCUCUCCCAAUUCUGCACAA-3' (SEQ ID NO: 361); and A-3', 5'-AA-3', 5'-GAA-3', 5'-UGAA-3', 5'-AUGAA-3', 5'-AAUGAA-3', 5'-GAAUGAA- 3', 5'-CGAAUGAA-3', 5'-ACGAAUGAA- 3', 5'-GACGAAUGAA-3' (SEQ ID NO: 362), 5'-AGACGAAUGAA-3' (SEQ ID NO: 363), 5'-UAGACGAAUGAA-3' (SEQ ID NO: 364), 5'-AUAGACGAAUGAA-3' ( SEQ ID NO: 365), 5'-AAUAGACGAAUGAA-3' (SEQ ID NO: 366), 5'-GAAUAGACGAAUGAA-3' (SEQ ID NO: 367), 5'-CGAAUAGACGAAUGAA-3' (SEQ ID NO: 368), 5'-CCGAAUAGACGAAUGAA-3 '(SEQ ID NO: 369), 5'-CCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 370), 5'-ACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 371), 5'-AACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 372), 5'-GAACCCGAAUAGACGAAUGAA -3' (SEQ ID NO: 373), 5'-AGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 374), 5'-CAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 375), 5'-GCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 376), 5' -UGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 377), 5'-UUGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 378), and 5'-GUUGCAGAACCCGAAUAGACGAAUGAA-3' (SEQ ID NO: 379) may additionally include a 13th sequence selected from the group consisting of have. In this case, the 3' end of the sixth sequence and the 5' end of the linker are connected through the twelfth sequence, and the 3' end of the linker and the 5' end of the seventh sequence are connected through the thirteenth sequence may have been

For example, when the twelfth sequence is 5'-U-3', the thirteenth sequence may be 5'-A-3'. As another example, when the twelfth sequence is 5'-UU-3', the thirteenth sequence may be 5'-AA-3'. As another example, when the twelfth sequence is 5'-UUC-3', the thirteenth sequence may be 5'-GAA-3'. As another example, when the twelfth sequence is 5'-UUCA-3', the thirteenth sequence may be 5'-UGAA-3'. As another example, when the twelfth sequence is 5'-UUCAU-3', the thirteenth sequence may be 5'-AUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUU-3', the thirteenth sequence may be 5'-AAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUU-3', the thirteenth sequence may be 5'-GAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUU-3', the thirteenth sequence may be 5'-CGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUU-3', the thirteenth sequence may be 5'-ACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUUC-3', the thirteenth sequence may be 5'-GACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUUCC-3', the thirteenth sequence may be 5'-AGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCU-3', the thirteenth sequence may be 5'-UAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUC-3', the thirteenth sequence may be 5'-AUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCU-3', the thirteenth sequence may be 5'-AAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUC-3', the thirteenth sequence may be 5'-GAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCC-3', the thirteenth sequence may be 5'-CGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCA-3', the thirteenth sequence may be 5'-CCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAA-3', the thirteenth sequence may be 5'-CCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAU-3', the thirteenth sequence may be 5'-ACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUU-3', the thirteenth sequence may be 5'-AACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUC-3', the thirteenth sequence may be 5'-GAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCU-3', the thirteenth sequence may be 5'-AGAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCUG-3', the thirteenth sequence may be 5'-CAGAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCUGC-3', the thirteenth sequence may be 5'-GCAGAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCUGCA-3', the thirteenth sequence may be 5'-UGCAGAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCUGCAC-3', the thirteenth sequence may be 5'-UUGCAGAACCCGAAUAGACGAAUGAA-3'. As another example, when the twelfth sequence is 5'-UUCAUUUUUCCUCUCCAAUUCUGCACA-3', or 5'-UUCAUUUUUCCUCUCCAAUUCUGCACAA-3', the thirteenth sequence may be 5'-GUUGCAGAACCCGAAUAGACGAAUGAA-3'.

Examples of engineered single guide RNA sequences

In one embodiment, the engineered single guide RNA consists of SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to SEQ ID NO: 407, SEQ ID NO: 409 to SEQ ID NO: 421, and SEQ ID NO: 436 to SEQ ID NO: 439 It may have a sequence selected from the group.

Engineered Dual Guide RNA Example 1

In one embodiment, the engineered Cas12f1 guide RNA comprises an engineered scaffold region, and a spacer.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region comprises the following sequence:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), a sequence selected from the group consisting of 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9),

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order,

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11), and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), 5'-AACAAAUUCAU- an engineered tracrRNA to which a sequence selected from the group consisting of 3' (SEQ ID NO: 67), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), and 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 12) is linked; and

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', 5' -AAUGAAGGA-3', and a sequence selected from the group consisting of 5'-GAAUGAAGGA-3' (SEQ ID NO: 14), and

5'-AUGCAAC-3' linked engineered crRNA repeat sequence portion,

At this time, the 3' end of the engineered crRNA repeat sequence portion is connected to the 5' end of the spacer.

At this time, the sequence of the engineered tracrRNA is 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAAUACCCUCGAAACAAAUUCAUUU-3' (sequence number 1) and different from the engineered crRNA (SEQ ID NO: 1) do.

For example, the sequence of the engineered tracrRNA may be the same as SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be different from SEQ ID NO: 3. As another example, the sequence of the engineered tracrRNA may be different from SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be the same as SEQ ID NO: 3. As another example, the sequence of the engineered tracrRNA may be different from SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be different from SEQ ID NO: 3.

For example, when the engineered tracrRNA includes 5'-AACAAA-3', the engineered crRNA may include 5'-GGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAU-3', the engineered crRNA may include 5'-AGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUU-3', the engineered crRNA may include 5'-AAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUC-3', the engineered crRNA may include 5'-GAAGGA-3'. As another example, when the engineered tracrRNA comprises 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), the engineered crRNA may include 5'-UGAAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUCAU-3' (SEQ ID NO: 67), the engineered crRNA may include 5'-AUGAAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), the engineered crRNA may include 5'-AAUGAAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 12), the engineered crRNA may include 5'-GAAUGAAGGA-3' (SEQ ID NO: 14).

Engineered Dual Guide RNA Example 2

In one embodiment, the engineered Cas12f1 guide RNA comprises an engineered scaffold region, and a spacer.

The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence.

The sequence of the engineered scaffold region comprises the following sequence:

5' end to 3' end direction,

5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5' -UGGAGAA-3', 5'-GUGGAGAA-3', 5'-AGUGGAGAA-3', 5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5' -UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), a sequence selected from the group consisting of 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9),

5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order,

5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11), and

5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), 5'-AACAAAUUCAU- 3' (SEQ ID NO: 67), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 69), 5'-AACAAAUUCAUUUU-3' (SEQ ID NO: 70), 5'- AACAAAUUCAUUUUU-3' (SEQ ID NO: 71), 5'-AACAAAUUCAUUUUUC-3' (SEQ ID NO: 72), 5'-AACAAAUUCAUUUUUCC-3' (SEQ ID NO: 73), 5'-AACAAAUUCAUUUUUUCCU-3' (SEQ ID NO: 74), 5 '-AACAAAUUCAUUUUUCCUC-3' (SEQ ID NO: 75), 5'-AACAAAUUCAUUUUUCCUCU-3' (SEQ ID NO: 76), 5'-AACAAAUUCAUUUUUCCUCUC-3' (SEQ ID NO: 77), 5'-AACAAAUUCAUUUUUUCCUCUCC-3' (SEQ ID NO: 78) , 5'-AACAAAUUCAUUUUUCCUCUCCA-3' (SEQ ID NO: 79), 5'-AACAAAUUCAUUUUUUCCUCUCCAA-3' (SEQ ID NO: 80), 5'-AACAAAUUCAUUUUUCCUCUCCAAU-3' (SEQ ID NO: 81), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUUUCCUCUCCAA 82), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUC-3' (SEQ ID NO: 83), 5'-AACAAAUUCAUUUUUUCCUCCCAAUUCU-3' (SEQ ID NO: 84), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCAUUCCUUCCAAUUCUG-3' (SEQ ID NO: 85), 5'-AACAA SEQ ID NO: 86), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCA-3' (SEQ ID NO: 87), 5'-AAACAAAUUCAUUUUUCCUCCCAAUUCUGCAC-3' (SEQ ID NO: 88), 5'-AAACAAAUUCAUUUUUCCUCUCCAAUUCUGCAUCA-3') (SEQ ID NO: an engineered tracrRNA to which a sequence selected from the group consisting of CAUUUUUCCUCCUCCAAUUCUGCACAA-3' (SEQ ID NO: 13) is linked; and

5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', 5' -AAUGAAGGA-3', 5'-GAAUGAAGGA-3' (SEQ ID NO: 90), 5'-CGAAUGAAGGA-3' (SEQ ID NO: 91), 5'-ACGAAUGAAGGA-3' (SEQ ID NO: 92), 5'-GACGAAUGAAGGA- 3' (SEQ ID NO: 93), 5'-AGACGAAUGAAGGA-3' (SEQ ID NO: 94), 5'-UAGACGAAUGAAGGA-3' (SEQ ID NO: 95), 5'-AUAGACGAAUGAAGGA-3' (SEQ ID NO: 96), 5'- AAUAGACGAAUGAAGGA-3' (SEQ ID NO: 97), 5'-GAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 98), 5'-CGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 99), 5'-CCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 100), 5 '-CCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 101), 5'-ACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 102), 5'-AACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 103), 5'-GAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 104) , 5'-AGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 105), 5'-CAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 106), 5'-GCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 107), 5'-UGCAGAACCCGAAUAGACGAAUGAAGGA-3' 108), 5'-UUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 109), and 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 15), and

5'-AUGCAAC-3' linked engineered crRNA repeat sequence portion,

At this time, the 3' end of the engineered crRNA repeat sequence portion is connected to the 5' end of the spacer.

이때, 상기 엔지니어링 된 tracrRNA의 서열은 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3'(서열번호 2)과 상이하고, 및/또는 상기 엔지니어링 된 crRNA 반복 서열 부분은 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3'(서열번호 4)과 상이 do.

For example, the sequence of the engineered tracrRNA may be the same as SEQ ID NO: 2, and the engineered crRNA repeat sequence portion may be different from SEQ ID NO: 4. As another example, the engineered tracrRNA sequence may be different from SEQ ID NO: 2, and the engineered crRNA repeat sequence portion may be identical to SEQ ID NO: 4. As another example, the engineered tracrRNA sequence may be different from SEQ ID NO: 2, and the engineered crRNA repeat sequence portion may be different from SEQ ID NO: 4.

For example, when the engineered tracrRNA includes 5'-AACAAA-3', the engineered crRNA may include 5'-GGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAU-3', the engineered crRNA may include 5'-AGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUU-3', the engineered crRNA may include 5'-AAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUC-3', the engineered crRNA may include 5'-GAAGGA-3'. As another example, when the engineered tracrRNA comprises 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), the engineered crRNA may include 5'-UGAAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUCAU-3' (SEQ ID NO: 67), the engineered crRNA may include 5'-AUGAAGGA-3'. As another example, when the engineered tracrRNA includes 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), the engineered crRNA may include 5'-AAUGAAGGA-3'. As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 69), the engineered crRNA may include 5'-GAAUGAAGGA-3' (SEQ ID NO: 90). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUU-3' (SEQ ID NO: 70), the engineered crRNA may include 5'-CGAAUGAAGGA-3' (SEQ ID NO: 91). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUU-3' (SEQ ID NO: 71), the engineered crRNA may include 5'-ACGAAUGAAGGA-3' (SEQ ID NO: 92). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUC-3' (SEQ ID NO: 72), the engineered crRNA may include 5'-GACGAAUGAAGGA-3' (SEQ ID NO: 93). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCC-3' (SEQ ID NO: 73), the engineered crRNA may include 5'-AGACGAAUGAAGGA-3' (SEQ ID NO: 94). As another example, if the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUUCCU-3' (SEQ ID NO: 74), the engineered crRNA may include 5'-UAGACGAAUGAAGGA-3' (SEQ ID NO: 95). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUC-3' (SEQ ID NO: 75), the engineered crRNA may include 5'-AUAGACGAAUGAAGGA-3' (SEQ ID NO: 96). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCU-3' (SEQ ID NO: 76), the engineered crRNA may include 5'-AAUAGACGAAUGAAGGA-3' (SEQ ID NO: 97). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUC-3' (SEQ ID NO: 77), the engineered crRNA may include 5'-GAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 98). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCC-3' (SEQ ID NO: 78), the engineered crRNA may include 5'-CGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 99). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCA-3' (SEQ ID NO: 79), the engineered crRNA may include 5'-CCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 100). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAA-3' (SEQ ID NO: 80), the engineered crRNA may include 5'-CCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 101). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAU-3' (SEQ ID NO: 81), the engineered crRNA may include 5'-ACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 102). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUU-3' (SEQ ID NO: 82), the engineered crRNA may include 5'-AACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 103). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUC-3' (SEQ ID NO: 83), the engineered crRNA may include 5'-GAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 104). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCU-3' (SEQ ID NO: 84), the engineered crRNA may include 5'-AGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 105). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUG-3' (SEQ ID NO: 85), the engineered crRNA may include 5'-CAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 106). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGC-3' (SEQ ID NO: 86), the engineered crRNA may include 5'-GCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 107). As another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCA-3' (SEQ ID NO: 87), the engineered crRNA may include 5'-UGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 108). As another example, when the engineered tracrRNA comprises 5'-AAACAAAUUCAUUUUUCCUCUCCCAAUUCUGCAC-3' (SEQ ID NO: 88), the engineered crRNA may include 5'-UUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 109). In another example, when the engineered tracrRNA comprises 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACA-3' (SEQ ID NO: 89) or 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 13), the engineered crRNA is 5'-GUUGACGAAUGAACCCGAAUUGACGAAUGAACCC ' (SEQ ID NO: 15).

Engineered CRISPR/Cas12f1 complex

Overview of the CRISPR/Cas12f1 complex

Provided herein is an engineered CRISPR/Cas12f1 complex. The engineered CRISPR/Cas12f1 complex comprises a Cas12f1 protein and an engineered Cas12f1 guide RNA. In this case, the engineered Cas12f1 guide RNA is as described in the "Engineered Cas12f1 guide RNA" section.

In one embodiment, the present specification provides an engineered CRISPR/Cas12f1 complex capable of editing a nucleic acid comprising a target sequence, including a Cas12f1 protein and an engineered Cas12f1 guide RNA. In this case, the engineered Cas12f1 guide RNA may be any one of those described in the "Engineered Cas12f1 guide RNA" paragraph.

Cas12f1 Protein - Overview

The engineered CRISPR/Cas12f1 complex provided herein includes a Cas12f1 protein. Basically, the Cas12f1 protein may be a wild-type Cas12f1 protein existing in nature. The sequence encoding the Cas12f1 protein may be a human codon-optimized Cas12f1 sequence for the wild-type Cas12f1 protein. In addition, the Cas12f1 protein may have the same function as a wild-type Cas12f1 protein existing in nature. However, unless specifically limited, as used herein, when the term "Cas12f1 protein" is used, it may encompass not only a wild-type or codon-optimized Cas12f1 protein, but also a modified Cas12f1 protein to a Cas12f1 fusion protein. In addition, as well as having the same function as the wild-type Cas12f1 protein existing in nature, all or part of the function is modified, all or part of the function is lost, and/or additional function is added. can The meaning of the Cas12f1 protein can be appropriately interpreted according to the context, and is interpreted in the broadest sense unless there is a special case. Hereinafter, the composition or function of the Cas12f1 protein will be described in detail.

Cas12f1 protein - wild-type Cas12f1 protein

The engineered CRISPR/Cas12f1 complex provided herein may include a Cas12f1 protein.

In one embodiment, the Cas12f1 protein may be a wild-type Cas12f1 protein. In one embodiment, the Cas12f1 protein may be derived from the Cas14 family (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)). In one embodiment, the Cas12f1 protein may be an uncultured archaeon-derived Cas14a protein (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)). In one embodiment, the Cas12f1 protein may be a Cas14a1 protein.

Cas12f1 protein - modified Cas12f1 protein

The engineered CRISPR/Cas12f1 complex provided herein may comprise a modified Cas12f1 protein. The modified Cas12f1 means that at least a portion of the wild-type or codon-optimized Cas12f1 protein sequence is modified. The Cas12f1 protein modification may be composed of individual amino acid units or may be composed of functional domain units of a protein.

In one embodiment, the modification of the protein may be one or more amino acids, peptides, polypeptides, proteins, and/or domains individually substituted, removed, and/or added in the wild-type or codon-optimized Cas12f1 protein sequence. . In one embodiment, the Cas12f1 protein may be one in which one or more amino acids, peptides, and/or polypeptides in the RuvC domain included in the wild-type Cas12f1 protein are substituted, removed, and/or added.

Cas12f1 protein - Cas12f1 fusion protein

The engineered CRISPR/Cas12f1 complex provided herein may comprise a Cas12f1 fusion protein. In this case, the Cas12f1 fusion protein refers to a protein in which an additional amino acid, peptide, polypeptide, protein, and/or domain is fused to a wild-type or modified Cas12f1 protein.

In one embodiment, the Cas12f1 protein may be a fusion of a base editor and/or reverse transcriptase to a wild-type Cas12f1 protein. In one embodiment, the base editor may be adenosine deaminase, and/or cytidine deaminase. In one embodiment, the reverse transcriptase may be Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, and/or a variant thereof. In this case, the Cas12f1 protein fused with the reverse transcriptase may function as a prime editor.

In one embodiment, the Cas12f1 protein may be a wild-type Cas12f1 protein fused with various enzymes that may be involved in the gene expression process in a cell. In this case, the Cas12f1 protein fused with the enzyme may cause various quantitative and qualitative changes in gene expression in cells. In one embodiment, the enzyme may be VP64, DNMT, TET, KRAB, DHAC, LSD, and/or p300.

Cas12f1 protein - alteration of function

The Cas12f1 protein included in the engineered CRISPR/Cas12f1 complex provided herein may have the same function as the wild-type Cas12f1 protein. The Cas12f1 protein included in the engineered CRISPR/Cas12f1 complex provided herein may have an altered function when compared to the wild-type Cas12f1 protein. Specifically, the alteration may be a modification of all or some functions, loss of all or some functions, and/or addition of additional functions. In one embodiment, the Cas12f1 protein is not particularly limited as long as it is a change that a person skilled in the art can apply to the Cas protein of the CRISPR/Cas system. In this case, the change may be made using a known technique.

In one embodiment, the Cas12f1 protein may be modified to cut only one of the double strands of the target nucleic acid. Furthermore, the Cas12f1 protein may be modified so that only one strand of the double strands of the target nucleic acid can be cut, and base editing or prime editing can be performed on the uncleaved strand. In one embodiment, the Cas12f1 protein may be modified so that it cannot cut all double-stranded strands of the target nucleic acid. Furthermore, the Cas12f1 protein cannot cut all double-strands of the target nucleic acid, and may be modified to perform a function of base editing, prime editing, or gene expression regulation for the target nucleic acid. .

Cas12f1 protein - examples of other modifications

In one embodiment, the Cas12f1 protein may include a Nuclear Localization Sequence (NLS) or a Nuclear Export Sequence (NES). Specifically, the NLS may be any one of those exemplified in the NLS paragraph of “Definitions of Terms”, but is not limited thereto. In one embodiment, the Cas12f1 protein may include a tag. Specifically, the tag may be any one of those exemplified in the tag paragraph of “Definition of Terms”, but is not limited thereto.

Cas12f1 protein - PAM sequence

Two conditions are required for the CRISPR/Cas12f1 complex to cleave a target gene or target nucleic acid. First, there must be a nucleotide sequence of a certain length that can be recognized by the Cas12f1 protein in the target gene or target nucleic acid. Second, there should be a sequence capable of complementary binding to the spacer sequence included in the guide RNA around the base sequence of the predetermined length. When the above two conditions are satisfied 1) the Cas12f1 protein recognizes the nucleotide sequence of the predetermined length, and 2) the spacer sequence portion complementarily binds to the sequence portion surrounding the nucleotide sequence of the predetermined length, a target gene, or The target nucleic acid is cleaved. In this case, the nucleotide sequence of a certain length recognized by the Cas12f1 protein is referred to as a Protospacer Adjacent Motif (PAM) sequence. The PAM sequence is a unique sequence determined according to the Cas12f1 protein, and when determining the target sequence of the CRISPR/Cas12f1 complex, there is a constraint that the target sequence must be determined within a sequence adjacent to the PAM sequence.

Cas12f1 protein - PAM sequence example

In one embodiment, the PAM sequence of the Cas12f1 protein may be a T-rich sequence. In one embodiment, the PAM sequence of the Cas12f1 protein may be THTN in the order from the 5' end to the 3' end. In this case, N is one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G), and H is deoxythymidine (T) , deoxyadenosine (A), and deoxycytidine (C). In one embodiment, the PAM sequence of the Cas12f1 protein may be TTTN in the order from the 5' end to the 3' end. In this case, N is one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G). In one embodiment, the PAM sequence of the Cas12f1 protein may be TTTA, TTTT, TTTC, or TTTG in the order from the 5' end to the 3' end. In one embodiment, the PAM sequence of the Cas12f1 protein may be TATA, TATT, TATC, or TATG in order from the 5' end to the 3' end. In one embodiment, the PAM sequence of the Cas12f1 protein may be TCTA, TCTT, TCTC, or TCTG in the order from the 5' end to the 3' end. In one embodiment, the PAM sequence of the Cas12f1 protein may be TTTA or TTTG in order from the 5' end to the 3' end. In one embodiment, the PAM sequence of the Cas12f1 protein may be different from the PAM sequence of the wild-type Cas12f1 protein.

Cas12f1 protein - sequence example

In one embodiment, the Cas12f1 protein may have an amino acid sequence selected from the group consisting of SEQ ID NO: 259 to SEQ ID NO: 266.

In one embodiment, the DNA sequence encoding the Cas12f1 protein may be a human codon-optimized sequence.

In one embodiment, the DNA sequence encoding the Cas12f1 protein may be a DNA sequence selected from the group consisting of SEQ ID NO: 267 to SEQ ID NO: 276.

Engineered Cas12f1 guide RNA

The engineered Cas12f1 guide RNA constituting the CRISPR/Cas12f1 complex provided herein has the same characteristics and structure as described in the section "Engineered Cas12f1 guide RNA".

CRISPR/Cas12f1 complex - construction example

In one embodiment, the Cas12f1 protein has an amino acid sequence selected from SEQ ID NO: 259 to SEQ ID NO: 262, and the engineered Cas12f1 guide RNA is SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to SEQ ID NO: 407, And it has a sequence selected from the group consisting of SEQ ID NO: 409 to SEQ ID NO: 421, and the Cas12f1 protein and the engineered Cas12f1 guide RNA bind to form a CRISPR/Cas12f1 complex.

In one embodiment, the Cas12f1 protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 263 to SEQ ID NO: 266, and the engineered Cas12f1 guide RNA is SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to sequence The CRISPR/Cas12f1 complex having a sequence selected from the group consisting of No. 407 and SEQ ID NO: 409 to SEQ ID NO: 421, wherein the Cas12f1 protein and the engineered Cas12f1 guide RNA are bound, may have a base editing function.

Vectors for expressing each component of the CRISPR/Cas12f1 system

vector outline

Provided herein are vectors for expressing components of the CRISPR/Cas12f1 system. The vector is configured to express a Cas12f1 protein, and/or an engineered Cas12f1 guide RNA. The sequence of the vector may include a nucleic acid sequence encoding one of the components of the CRISPR/Cas12f1 system, or may include a nucleic acid sequence encoding two or more components. The sequence of the vector comprises a nucleic acid sequence encoding a Cas12f1 protein and/or a nucleic acid sequence encoding an engineered Cas12f1 guide RNA. The sequence of the vector includes one or more promoter sequences. The promoter is operatively linked with a nucleic acid sequence encoding a Cas12f1 protein and/or a nucleic acid sequence encoding an engineered Cas12f1 guide RNA, such that transcription of the nucleic acid sequence in a cell can be promoted. The Cas12f1 protein has the same characteristics and composition as the Cas12f1 protein, the modified Cas12f1 protein, and/or the Cas12f1 fusion protein described in the section "Engineered CRISPR/Cas12f1 complex". The engineered Cas12f1 guide RNA has the same characteristics and configuration as the engineered Cas12f1 guide RNA described in the section "Engineered Cas12f1 guide RNA".

The sequence of the vector may include a nucleic acid sequence encoding a Cas12f1 protein and a nucleic acid sequence encoding an engineered Cas12f1 guide RNA. In one embodiment, the sequence of the vector may include a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein, and a second sequence comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA. The sequence of the vector comprises a promoter sequence for expressing a nucleic acid sequence encoding a Cas12f1 protein in a cell, and a promoter sequence for expressing a nucleic acid sequence encoding an engineered Cas12f1 guide RNA in a cell, wherein the promoters are each It is operably linked to the expression target. In one embodiment, the sequence of the vector may include a first promoter sequence operably linked to the first sequence, and a second promoter sequence operably linked to the second sequence.

The sequence of the vector may include a nucleic acid sequence encoding a Cas12f1 protein and a nucleic acid sequence encoding two or more different engineered Cas12f1 guide RNAs. In one embodiment, the sequence of the vector comprises a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein, a second sequence comprising a nucleic acid sequence encoding a first engineered Cas12f1 guide RNA, a second engineered Cas12f1 guide RNA It may include a third sequence comprising a nucleic acid sequence encoding Furthermore, the sequence of the vector comprises a first promoter sequence operably linked to the first sequence, a second promoter sequence operably linked to the second sequence, and a third promoter sequence operably linked to the third sequence may include

Expression target - Cas12f1 protein

The vector may be configured to express the Cas12f1 protein. In this case, the Cas12f1 protein has the same composition and characteristics as those described in the section "Engineered CRISPR/Cas12f1 complex".

In one embodiment, the vector may be configured to express a wild-type Cas12f1 protein. In this case, the wild-type Cas12f1 protein may be Cas14a1. In one embodiment, the vector may be configured to express the Cas12f1 protein modified to cut only one strand of the double strand of the target nucleic acid. Furthermore, the altered Cas12f1 protein can cut only one of the double strands of the target nucleic acid, and base editing or prime editing can be performed on the uncleaved strand. . In one embodiment, the Cas12f1 protein may be modified so that it cannot cut all double-stranded strands of the target nucleic acid. Furthermore, the Cas12f1 protein cannot cut all double-strands of the target nucleic acid, and may be modified to perform a function of base editing, prime editing, or gene expression regulation for the target nucleic acid. .

Expression target - engineered Cas12f1 guide RNA

The vector may be configured to express an engineered Cas12f1 guide RNA. The engineered Cas12f1 guide RNA has the same characteristics and configuration as the engineered Cas12f1 guide RNA described in the section "Engineered Cas12f1 guide RNA". The vector may be configured to express two or more different engineered Cas12f1 guide RNAs.

Expression Target - Additional Components

The vector may be configured to express additional components such as NLS and tag proteins in addition to the above-described expression target. In one embodiment, the additional component may be expressed independently of the Cas12f1, the modified Cas12f1, and/or the engineered Cas12f1 guide RNA. In another embodiment, the additional component may be expressed in connection with the Cas12f1, the modified Cas12f1, and/or the engineered Cas12f1 guide RNA. In this case, the additional component may be a component that is generally expressed when expressing the CRISPR/Cas system, and known techniques may be referred to. The additional component may be one or more of the components described in the section "Background - CRISPR/Cas system expression vector design".

Vector Construction - Cas12f1 protein expression sequence

The vector sequence may include a nucleic acid sequence encoding the Cas12f1 protein. In this case, the Cas12f1 protein has the same composition and characteristics as those described in the section "Engineered CRISPR/Cas12f1 complex".

In one embodiment, the sequence of the vector may include a sequence encoding a wild-type Cas12f1 protein. In this case, the wild-type Cas12f1 protein may be Cas14a1. In one embodiment, the sequence of the vector may include a human codon-optimized nucleic acid sequence encoding the Cas12f1 protein. In this case, the human codon-optimized nucleic acid sequence encoding the Cas12f1 protein may be a human codon-optimized nucleic acid sequence encoding the Cas14a1 protein. In one embodiment, the sequence of the vector may include a sequence encoding a modified Cas12f1 protein or a Cas12f1 fusion protein. In one embodiment, the sequence of the vector can cut only one strand among the double strands of the target nucleic acid, and the non-cleaved strand Cas12f1 is modified to perform base editing or prime editing. It may include a sequence encoding a protein. In one embodiment, the Cas12f1 protein cannot cut all double-strands of the target nucleic acid, and the Cas12f1 modified to function for base editing, prime editing, or gene expression regulation of the target nucleic acid. It may include a sequence encoding a protein.

Vector Construction - Engineered Cas12f1 Guide RNA Expression Sequence

In one embodiment, the sequence of the vector may include a sequence encoding the engineered Cas12f1 guide RNA. For example, the sequence of the vector is SEQ ID NO: 210 to SEQ ID NO: 258, SEQ ID NO: 381 to SEQ ID NO: 393, SEQ ID NO: 395 to SEQ ID NO: 407, SEQ ID NO: 409 to SEQ ID NO: 421, and SEQ ID NO: 436 to SEQ ID NO: 439 It may comprise a sequence selected from the group consisting of.

In one embodiment, the sequence of the vector may include sequences encoding two or more different engineered Cas12f1 guide RNAs. For example, the sequence of the vector is SEQ ID NO: 210 to SEQ ID NO: 258, SEQ ID NO: 381 to SEQ ID NO: 393, SEQ ID NO: 395 to SEQ ID NO: 407, SEQ ID NO: 409 to SEQ ID NO: 421, and SEQ ID NO: 436 to SEQ ID NO: 439 It may include a sequence encoding a first engineered Cas12f1 guide RNA, each selected from the group consisting of, and a sequence encoding a second engineered Cas12f1 guide RNA.

Vector Construction - Promoter Sequence

The vector sequence includes a promoter sequence operably linked to a sequence encoding each element. Specifically, the promoter sequence may be, but is not limited to, one of the promoters disclosed in the promoter part of the "Background - CRISPR/Cas system expression vector design" section.

In one embodiment, the vector sequence may include a sequence encoding a Cas12f1 protein, and a promoter sequence. In this case, the promoter sequence is operably linked to the sequence encoding the Cas12f1 protein. In one embodiment, the vector sequence may include a sequence encoding the engineered Cas12f1 guide RNA, and a promoter sequence. In this case, the promoter sequence is operably linked with the sequence encoding the engineered Cas12f1 guide RNA. In one embodiment, the vector sequence may include a sequence encoding a Cas12f1 protein, a sequence encoding an engineered Cas12f1 guide RNA, and a promoter sequence. In this case, the promoter sequence is operatively linked with the sequence encoding the Cas12f1 protein and the engineered Cas12f1 guide RNA, and the transcription factor activated due to the promoter sequence is the Cas12f1 protein and the engineered Cas12f1 Guide RNA is expressed.

Vector construction - can contain more than one promoter sequence

In one embodiment, the vector sequence may include a first sequence encoding a Cas12f1 protein, a first promoter sequence, a second sequence encoding an engineered Cas12f1 guide RNA, and a second promoter sequence. In this case, the first promoter sequence is operably linked to the first sequence, the second promoter sequence is operatively linked to the upper limb second sequence, and transcription of the first sequence is performed by the first promoter sequence. induced, and transcription of the second sequence is induced by the second promoter sequence. In this case, the first promoter and the second promoter may be the same type of promoter. In this case, the first promoter and the second promoter may be different types of promoters.

In one embodiment, the vector sequence comprises a first sequence encoding a Cas12f1 protein, a first promoter sequence, a second sequence encoding a first engineered Cas12f1 guide RNA, a second promoter sequence, and a second engineered Cas12f1 guide RNA. a third sequence encoding, and a third promoter sequence. wherein the first promoter sequence is operatively linked with the first sequence, the second promoter sequence is operably linked with the second sequence, and the third promoter sequence is operably linked with the third sequence. linked, wherein transcription of the first sequence is induced by the first promoter sequence, transcription of the second sequence is induced by the second promoter sequence, and transcription of the third sequence is induced by the third promoter sequence is induced In this case, the second promoter and the third promoter may be the same type of promoter. Specifically, the second promoter sequence and the third promoter sequence may be a U6 promoter sequence, but is not limited thereto. In this case, the second promoter and the third promoter may be different types of promoters. Specifically, the second promoter may be a U6 promoter sequence, and the third promoter may be a H1 promoter sequence, but is not limited thereto.

Vector Construction - Termination Signal

The vector may include a termination signal operably linked to the promoter sequence. In this case, the termination signal may be one of the termination signals disclosed in the termination signal portion of the "Background - CRISPR/Cas system expression vector design" section, but is not limited thereto. The termination signal may vary depending on the type of promoter sequence.

In one embodiment, when the vector sequence includes a U6 promoter sequence, a thymidine sequence operably linked to the U6 promoter sequence may serve as a termination signal. In one embodiment, the thymidine sequence may be a sequence in which 5 or more thymidine is continuously linked. In one embodiment, when the vector sequence includes an H1 promoter sequence, a thymidine sequence operably linked to the H1 promoter sequence may serve as a termination signal. In one embodiment, the thymidine sequence may be a sequence in which 5 or more thymidine is continuously linked.

Vector Composition - Miscellaneous Composition

The vector sequence may include elements necessary according to the purpose in addition to the above construction.

In one embodiment, the vector sequence may include regulatory/control element sequences, and/or additional element sequences. In one embodiment, the additional component may be added for the purpose of distinguishing the transfected cells from the non-transfected cells. In this case, the regulatory/control element sequence and additional element may be one of those disclosed in the "Background - CRISPR/Cas system expression vector design" section, but is not limited thereto.

Vector Type - Virus Vector

The vector may be a viral vector.

In one embodiment, the viral vector may be one or more selected from the group consisting of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vacciniaviruses, poxviruses, and herpes simplex viruses. In one embodiment, the viral vector may be an adeno-associated virus.

Vector type - non-viral vector

The vector may be a non-viral vector. In one embodiment, the non-viral vector may be one or more selected from the group consisting of plasmids, phages, naked DNA, DNA complexes, and mRNA. In one embodiment, the plasmid is a pcDNA series, pSC101, pG1796, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. It may be selected from the group consisting of. In one embodiment, the phage may be selected from the group consisting of λgt4λB, λ-Charon, λΔz1, and M13. In one embodiment, the vector may be a PCR amplicon.

Vector form - circular or linear vector

The vector may have a circular or linear form. When the vector is a linear vector, RNA transcription is terminated at its 3' end even if the linear vector sequence does not separately include a termination signal. In comparison, when the vector is a circular vector, RNA transcription is not terminated unless the circular vector sequence separately includes a termination signal. Therefore, when a circular vector is used as the vector, a termination signal corresponding to a transcription factor related to each promoter sequence should be included in order to express an intended target.

In one embodiment, the vector may be a linear vector. In one embodiment, the vector may be a linear amplicon. In one embodiment, the vector comprises a sequence selected from SEQ ID NO: 267 to SEQ ID NO: 276 and SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to SEQ ID NO: 407, and SEQ ID NO: 409 to SEQ ID NO: 421 It may be a linear vector comprising a sequence selected from In one embodiment, the vector may be a circular vector. In one embodiment, the vector consists of a sequence selected from SEQ ID NO: 267 to SEQ ID NO: 276, and SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to SEQ ID NO: 407, and SEQ ID NO: 409 to SEQ ID NO: 421 It may be a circular vector comprising a sequence selected from the group.

Vector - Sequence Example

In one embodiment, the sequence of the vector comprises a sequence selected from SEQ ID NO: 267 to SEQ ID NO: 276, and SEQ ID NO: 210 to 258, SEQ ID NO: 381 to 393, SEQ ID NO: 396 to SEQ ID NO: 407, and SEQ ID NO: 409 to SEQ ID NO: 421 It may include a sequence selected from the group consisting of.

chemical modification of nucleic acids

In the present specification, an engineered crRNA or a nucleic acid encoding the same, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and/or a vector for expressing a component of the CRISPR/Cas12f1 system. to provide. In this case, the term "nucleic acid" in the above component may be DNA or RNA existing in nature, and may be a modified nucleic acid in which some or all of the component nucleic acid is chemically modified. In one embodiment, the constituent nucleic acid may be DNA and/or RNA existing in nature. In one embodiment, the constituent nucleic acid may be one in which one or more nucleotides are chemically modified. In this case, the chemical modification includes all modifications of nucleic acids known to those skilled in the art. Specifically, the chemical modification may include all modifications of the nucleic acid described in (WO 2019/089820 A1), but is not limited thereto.

Gene editing method using engineered Cas12f1 guide RNA

Overview of Gene Editing Methods

The present specification provides a method for editing a target gene or target nucleic acid in a target cell using an engineered crRNA. The target gene, or target nucleic acid, includes a target sequence. The target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. The gene editing method includes delivering an engineered Cas12f1 guide RNA, a Cas12f1 protein, or a nucleic acid encoding each of the target gene or target nucleic acid into a target cell. As a result, the engineered CRISPR/Cas12f1 complex is injected into the target cell, or the engineered CRISPR/Cas12f1 complex is induced, and the target gene is edited by the engineered CRISPR/Cas12f1 complex. The engineered Cas12f1 guide RNA has the same characteristics and structure as described in the section "Engineered Cas12f1 guide RNA". The Cas12f1 protein has the same characteristics and composition as the Cas12f1 protein described in the section "Engineered CRISPR/Cas12f1 complex", and/or the modified Cas12f1 protein.

In one embodiment, the gene editing method may include delivering a Cas12f1 protein or a nucleic acid encoding the same, and an engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a target cell.

At this time, the engineered Cas12f1 guide RNA includes an engineered scaffold region, and a spacer.

In this case, the engineered scaffold region has the same characteristics and structure as described in any one of the above-mentioned “engineered scaffold region” paragraphs. For example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOs: 186 to 167. As another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOs: 187 to 198. As another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOs: 199 to 205. As another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOs: 206 to 209.

In this case, the spacer sequence may complementarily bind to a target gene or target nucleic acid included in the target cell.

target cell

In one embodiment, the target cell may be a prokaryotic cell. In one embodiment, the target cell may be a eukaryotic cell. Specifically, the eukaryotic cell may be, but is not limited to, a plant cell, an animal cell, and/or a human cell.

target sequencing

The target gene, or target nucleic acid, and target sequence to be edited with the CRISPR/Cas12f1 complex may be determined in consideration of the purpose of gene editing, the target cell environment, the PAM sequence recognized by the Cas12f1 protein, and/or other variables. In this case, if the target sequence of an appropriate length can be determined, the method is not particularly limited, and a known technique may be used.

Determination of spacer sequence according to target sequence

After the target sequence is determined, a corresponding spacer sequence is designed. The spacer sequence is designed as a sequence capable of complementary binding to the target sequence. In one embodiment, the spacer sequence is designed as a sequence capable of complementary binding to the target gene. In one embodiment, the spacer sequence is designed to complementarily bind to the target nucleic acid. In one embodiment, the spacer sequence is designed as a sequence complementary to a target sequence included in the target strand sequence of the target nucleic acid. In one embodiment, the spacer sequence is designed as an RNA sequence corresponding to the DNA sequence of the protospacer included in the non-target strand sequence of the target nucleic acid. Specifically, the spacer sequence has the same nucleotide sequence as the protospacer sequence, but is designed as a sequence in which all thymidine included in the nucleotide sequence are substituted with uridine.

Complementarity of target sequence and spacer sequence

In one embodiment, the spacer sequence and the target sequence 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary sequence. In one embodiment, the spacer sequence may be a sequence complementary to the target sequence within a numerical range selected in the immediately preceding sentence. For example, the spacer sequence may be a sequence that is 60% to 90% complementary to the target sequence. As another example, the spacer sequence may be a sequence that is 90% to 100% complementary to the target sequence.

Number of mismatches between target sequence and spacer sequence

In one embodiment, the spacer sequence has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches with the target sequence. It may be a complementary sequence. In one embodiment, the spacer sequence may have a mismatch within the numerical range selected in the immediately preceding sentence. For example, the spacer sequence may have 1 to 5 mismatches with the target sequence. As another example, the spacer sequence may substrate 6 to 10 mismatches with the target sequence.

Using the CRISPR/Cas12f1 complex

The gene editing method provided herein utilizes the fact that the engineered CRISPR/Cas12f1 complex has target-specific gene or nucleic acid cleavage activity. The engineered CRISPR/Cas12f1 complex has the same characteristics and composition as the engineered CRISPR/Cas12f1 complex described in the section "Engineered CRISPR/Cas12f1 complex".

Intracellular delivery of each component of the CRISRP/Cas12f1 complex

The gene editing method provided herein is based on the premise that the engineered CRISPR/Cas12f1 complex in a target cell contacts a target gene or target nucleic acid. Therefore, in order to induce the engineered CRISPR/Cas12f1 complex to contact the target gene or target nucleic acid, the gene editing method comprises delivering each component of the engineered CRISPR/Cas12f1 complex into a target cell. . In one embodiment, the gene editing method may include delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same and a Cas12f1 protein or a nucleic acid encoding the same into a target cell. In one embodiment, the gene editing method may include delivering engineered Cas12f1 guide RNA and Cas12f1 protein into a target cell. In one embodiment, the gene editing method may include delivering a nucleic acid encoding an engineered Cas12f1 guide RNA and a Cas12f1 protein into a target cell. In one embodiment, the gene editing method may include delivering an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein into a target cell. In one embodiment, the gene editing method may include delivering a nucleic acid encoding an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein into a target cell. The engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and the Cas12f1 protein or a nucleic acid encoding the same may be delivered into a target cell in various delivery forms and using various delivery methods.

Delivery mode - RNP

As the delivery form, engineered Cas12f1 guide RNA and ribonucleoprotein particle (RNP) to which Cas12f1 protein is bound can be used. In one embodiment, the gene editing method may include injecting a CRISPR/Cas12f1 complex to which an engineered Cas12f1 guide RNA and Cas12f1 protein are bound into a target cell.

Mode of delivery - non-viral vector

As another form of delivery, a non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein may be used. In one embodiment, the gene editing method may include injecting a non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein into a target cell. Specifically, the non-viral vector may be a plasmid, naked DNA, DNA complex, or mRNA, but is not limited thereto. In another embodiment, the gene editing method comprises a first non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA, and a second non-viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein into a target cell. This may include injecting. Specifically, the first non-viral vector and the second non-viral vector may each be one selected from the group consisting of plasmid, naked DNA, DNA complex, and mRNA, but is not limited thereto.

Transmission mode - viral vector

As another form of delivery, a viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein may be used. In one embodiment, the gene editing method may include injecting a viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein into a target cell. Specifically, the viral vector may be one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus and herpes simplex virus, but is not limited thereto. In one embodiment, the viral vector may be an adeno-associated virus.

In another embodiment, the gene editing method comprises injecting a first viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA, and a second viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein into a target cell. may include Specifically, the first viral vector and the second viral vector may be one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus, respectively. It is not limited.

Method of delivery - common means of delivery

The delivery method is not particularly limited as long as it can deliver the Cas12f1 guide RNA engineered into a cell or a nucleic acid encoding the same, and the Cas12f1 protein or a nucleic acid encoding the same into a cell in an appropriate delivery form. In one embodiment, the delivery method may be electroporation, gene gun, sonoporation, magnetofection, and/or transient cell compression or squeezing.

Delivery Method - Nanoparticles

The delivery method may be to deliver at least one component included in the CRISPR/Cas12f1 system using nanoparticles. In this case, the delivery method may be a known method that can be appropriately selected by those skilled in the art. For example, the nanoparticle delivery method may be a method disclosed in (WO 2019/089820 A1), but is not limited thereto.

In one embodiment, the delivery method may be to deliver a Cas12f1 protein or a nucleic acid encoding the same and/or an engineered Cas12f1 guide RNA or a nucleic acid encoding the same using nanoparticles. In one embodiment, the delivery method comprises a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and/or a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same using nanoparticles. may be transmitted. At this time, the delivery method is cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation (precipitation), lipofection, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection , and/or nanoparticle-mediated nucleic acid delivery (see Panyam et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023) may be, but is not limited thereto. In this case, the components of the CRISPR/Cas12f1 system may be in the form of RNPs, non-viral vectors, and/or viral vectors. For example, the components of the CRISPR/Cas12f1 system may be in the form of mRNA encoding each component, but is not limited thereto.

Delivery form and method - Combination possible

The gene editing method includes intracellular delivery of an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same, wherein the delivery form and/or delivery method of the construct may be the same or different from each other. can In one embodiment, the gene editing method may include delivering the engineered Cas12f1 guide RNA or a nucleic acid encoding the same in a first delivery mode, and delivering the Cas12f1 protein or a nucleic acid encoding the same in a second delivery mode. In this case, each of the first delivery mode and the second delivery mode may be any one of the aforementioned delivery modes. In one embodiment, the gene editing method may include delivering the engineered Cas12f1 guide RNA or nucleic acid encoding the same by the first delivery method, and delivering the Cas12f1 protein or the nucleic acid encoding the same by the second delivery method. In this case, the first delivery method and the second delivery method may be any one of the aforementioned delivery methods, respectively.

delivery order

The gene editing method includes intracellular delivery of an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same, wherein the construct can be simultaneously delivered into the cell, but sequentially with a time difference can be transmitted.

In one embodiment, the gene editing method may include simultaneously delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same. In one embodiment, the gene editing method may include delivering the engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a cell, and then delivering the Cas12f1 protein or a nucleic acid encoding the same into the cell with a time difference. In one embodiment, the gene editing method may include delivering a Cas12f1 protein or a nucleic acid encoding the same into a cell, and then delivering an engineered Cas12f1 guide RNA into the cell with a time difference. In one embodiment, the gene editing method may include delivering the Cas12f1 protein-encoding nucleic acid into the cell, and then delivering the engineered Cas12f1 guide RNA into the cell with a time difference.

Delivery of multiple engineered Cas12f1 guide RNAs or nucleic acids encoding them

The gene editing method provided herein may include delivering a Cas12f1 protein or a nucleic acid encoding the same, and two or more engineered Cas12f1 guide RNAs or a nucleic acid encoding the same into a target cell. Through the above method, two or more CRISPR/Cas12f1 complexes targeting different sequences may be injected into or formed in a target cell. As a result, two or more target genes or target nucleic acids contained in the cell can be edited. In one embodiment, the gene editing method comprises a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a target gene or a target nucleic acid It includes delivery into the target cell containing the. In this case, each of the components may be delivered into a cell using one or more of the above-described delivery forms and delivery methods. In this case, two or more components may be simultaneously delivered into the cell, and may be sequentially delivered with a time difference.

CRISPR/Cas12f1 complex contacts target nucleic acid

In the gene editing method provided herein, editing of a target gene or target nucleic acid is performed while an engineered CRISPR/Cas12f1 complex in a target cell contacts the target gene or target nucleic acid. Accordingly, the gene editing method may include allowing the engineered CRISPR/Cas12f1 complex to contact within a target cell or inducing contact. In one embodiment, the gene editing method may include contacting the engineered CRISPR/Cas12f1 complex with a target nucleic acid in a target cell. In one embodiment, the gene editing method may include inducing the engineered CRISPR/Cas12f1 complex to contact a target nucleic acid in a target cell. At this time, the induction is not particularly limited as long as it is a method for allowing the engineered CRISPR/Cas12f1 complex to contact a target nucleic acid in a cell. In one embodiment, the induction may be to deliver an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same into a cell.

Gene Editing Results - Indels

As a result of performing the gene editing method provided herein, an indel may occur in a target gene or target nucleic acid. In this case, the indel may occur inside and/or outside the target sequence portion and/or the protospacer sequence portion. The indel refers to a mutation in which some nucleotides are deleted in the middle, arbitrary nucleotides are inserted, and/or the insertion and deletion are incorporated in the nucleotide sequence of the nucleic acid before gene editing. In general, when an indel occurs within a target gene or target nucleic acid sequence, the gene or nucleic acid is inactivated. In one embodiment, as a result of performing the gene editing method, one or more nucleotides in the target gene or target nucleic acid may be deleted and/or added.

Gene editing results - base editing

As a result of performing the gene editing method provided herein, base editing in a target gene or target nucleic acid may occur. This means that one or more specific bases in a nucleic acid are altered as intended, unlike indels in which any bases in the target gene or target nucleic acid are deleted or added. In other words, in a specific position in the target gene or target nucleic acid, it is to cause a point mutation (point mutation) intended in advance. In one embodiment, as a result of performing the gene editing method, one or more bases in the target gene or target nucleic acid may be substituted with another base.

Gene Editing Results - Insertion

As a result of performing the gene editing method provided herein, a knock-in in the target gene or target nucleic acid may occur. The knock-in refers to inserting an additional nucleic acid sequence into a target gene or target nucleic acid sequence. In order for the knock-in to occur, a donor comprising the additional nucleic acid sequence in addition to the CRISPR/Cas12f1 complex is further required. When the CRISPR/Cas12f1 complex cleaves a target gene or target nucleic acid in a cell, repair of the cleaved target gene or target nucleic acid occurs. In this case, the donor participates in the repair process so that the additional nucleic acid sequence can be inserted into the target gene or target nucleic acid. In one embodiment, the gene editing method may further comprise delivering a donor into a target cell. In this case, the donor includes the additional target nucleic acid, and insertion of the additional target nucleic acid into the target gene or the target nucleic acid is induced by the donor. In this case, when delivering the donor into a target cell, the above-described delivery form and/or delivery method may be used.

Gene Editing Results - Deletion

As a result of performing the gene editing method provided herein, all or part of the target gene or target nucleic acid sequence may be removed. The removal means removing a partial nucleotide sequence in the target gene or the target nucleic acid. In one embodiment, the gene editing method comprises a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a target gene or a target nucleic acid It includes introducing into a cell comprising a. Due to this, as a result of the gene editing, removal of a specific sequence portion in the target gene or target nucleic acid occurs.

Examples of gene editing methods

In one embodiment, the gene editing method may include delivering the CRISPR/Cas12f1 complex in the form of ribonucleoprotin particles to which the engineered Cas12f1 guide RNA and Cas12f1 protein are bound into a eukaryotic cell. In this case, the delivery may be using electroporation or lipofection.

In one embodiment, the gene editing method may include delivering a nucleic acid encoding an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein into a eukaryotic cell. In this case, the delivery may be using electroporation or lipofection.

In one embodiment, the gene editing method may comprise delivering an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein into a eukaryotic cell. .

In one embodiment, the gene editing method comprises adeno-association comprising a nucleic acid sequence encoding a first engineered Cas12f1 guide RNA, a nucleic acid sequence encoding a second engineered Cas12f1 guide RNA, and a nucleic acid sequence encoding a Cas12f1 protein and delivering a viral (AAV) vector into a eukaryotic cell.

Experimental example

Hereinafter, the invention provided by the present specification will be described in more detail through experimental examples and examples. These examples are only for illustrating the content disclosed by the present specification, and it is to those skilled in the art that the scope of the content disclosed by the present specification is not to be construed as being limited by these examples. it will be self-evident

Experimental Example 1 Preparation of test materials

Experimental Example 1.1 Plasmid Vector Design and Preparation

For expression in human cells, the Cas12f1 gene was codon-optimized, and the optimized sequence was synthesized for vector preparation. Finally, the sequence encoding the Cas12f1 protein included a chicken β-actin promoter, a nuclear localization signal sequence at the 5'- and 3'-ends, and a sequence encoding eGFP linked by a self-cleaving T2A peptide. The amino acid sequence of the Cas12f1 protein and the DNA sequence encoding it are shown in [Table 01].

[Table 01]

Template DNA encoding the (engineered) Cas12f1 guide RNA was synthesized and cloned into the pTwist Amp plasmid vector (Twist Bioscience). If necessary, the vector was used as a template for amplification of the guide RNA coding sequence using a U6-complementary forward primer and a protospacer-complementary reverse primer. Using Gibson assembly, the vector for the engineered CRISPR/Cas12f1 system was prepared by cloning the oligonucleotide encoding the engineered Cas12f1 guide RNA into a vector containing the codon-optimized Cas12f1 gene.

Experimental Example 1.2 Cas12f1 guide RNA engineering

Modifications of the second, fourth and fifth regions of the engineered scaffold region of the engineered Cas12f1 guide RNA were performed using ApoI and BamHI restriction enzymes to linearize the guide RNA-encoding vector with the modified sequence (Macrogen). This was done by cloning the synthetic oligonucleotides that delivered. Modification of the first region of the engineered scaffold region of the engineered Cas12f1 guide RNA canonical (canonical) using a forward primer targeting the 5' end of the tracrRNA and a reverse primer targeting the U6 promoter region ), or by PCR amplification of the engineered template plasmid vector. The PCR amplification was performed by Q5 Hot Start high-fidelity DNA polymerase (NEB), and the PCR product was ligated using KLD Enzyme Mix (NEB). The ligated PCR product was transformed into DH5α E. coli cells. Mutagenesis was confirmed by Sanger sequencing analysis. The modified plasmid vector was purified using NucleoBond ® Xtra Midi EF kit (MN). 1 microgram of purified plasmid was used as a template for mRNA synthesis using T7 RNA polymerase (NEB) and NTPs (Jena Bioscience). The engineered Cas12f1 guide RNA prepared above was purified using Monarch® RNA cleanup kit (NEB), aliquoted into cryogenic vials and stored in liquid nitrogen.

Experimental Example 1.3 Cell culture and transfection

HEK293 T cells (LentX-293T, Takara) were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) serum (Corning) and penicillin/streptomycin, 5% CO Incubated under ₂ conditions. Cell transfection was performed by electroporation or lipofection. In the case of electroporation, 2-5 μg of each DNA encoding the plasmid vector and guide RNA (and engineered guide RNA) encoding the Cas12f1 protein prepared in Experimental Example 1.2 were used with the Neon transfection system (Invitrogen) to 4X10 ⁵ HEK -293 T cells were transfected. The electroporation method was performed under 1300V, 10 mA, 3 pulse conditions. For lipofection, 6-15 μL FuGene reagent (Promega) was mixed with 2-5 μg of the plasmid vector encoding the Cas12f1 protein and 1.5-5 μg of the PCR amplicon for 15 min. The mixture (300 μL) was added to 1.5 ml DMEM medium plated with 1× ^{10 6} cells 1 day before transfection. The cells were cultured for 1 to 10 days in the presence of the mixture. After incubation, the cells were collected, and the genomic DNA of the cells was manually isolated using the PureHelix™ genomic DNA preparation kit (NanoHelix) or the Maxwell RSC Cultured cells DNA Kit (Promega).

Experimental Example 1.4 Measurement of Indel Efficiency in Cells

Among the genomic DNA isolated from HEK-293 T cells, the region including the protospacer was subjected to PCR in the presence of KAPA HiFi HotStart DNA polymerase (Roche) using target-specific primers. The amplification method followed the manufacturer's instructions. The PCR amplicon resulting from the amplification containing Illumina TruSeq HT dual indexes was subjected to 150-bp pair-end sequencing using Illumina iSeq 100. Indel frequencies were calculated using MAUND. The MAUND is provided at https://github.com/ibscge/maund.

Experimental Example 1.5 Quantitave real-time PCR

Guide RNA (or engineered guide RNA) or genomic DNA was extracted from HEK293 T cells using RNeasy Miniprep kit (Qiagen), Maxwell RSC miRNA Tissue Kit (Promega), or DNeasy Blood & Tissue Kit (Qiagen), respectively. To quantify guide RNA, RNA-specific primers were ligated, and cDNA was synthesized using crRNA-specific primers. The cDNA was used as a template for quantitative real-time PCR. Real-time PCR was analyzed using the KAFA SYBR FAST qPCR Master Mix (2X) Kit (KAPAbiosystems).

Experimental Example 1.6 Statistical Analysis

The experiment for each experimental example was performed three times, and the average value of each value was used for analysis.

Experimental Example 2 Comparison of indel efficiency of the engineered CRISPR/Cas12f1 system

In order to measure the indel efficiency of the engineered CRISPR/Cas12f1 system using the engineered Cas12f1 guide RNA, each Example was prepared by Experimental Examples 1.1 to 1.2. The target sequences used in the experiment are shown in Table 02 below.

[Table 02]

The sequences of the engineered Cas12f1 guide RNAs for each Example are shown in the following [Table 03] to [Table 08].

[Table 03]

[Table 04]

[Table 05]

[Table 06]

[Table 07]

[Table 08]

At this time, for each target sequence,

One) Comparative Example n.1 is a single guide RNA in which Cas12f1 tracrRNA found in nature and Cas12f1 crRNA found in nature are linked with 5'-GAAA-3';

2) Examples n.1 to n.3 are an engineered scaffold region having a modified first region, and an engineered Cas12f1 guide RNA having a spacer,

3) Examples n.4 to n.6 are an engineered scaffold region having a modified second region, and an engineered Cas12f1 guide RNA having a spacer,

4) Examples n.7 to n.9 show an engineered scaffold region having a modified fourth region and a fifth region, and an engineered Cas12f1 guide RNA having a spacer,

5) Example n.10 is an engineered scaffold region having a modified first region and a second region, and an engineered Cas12f1 guide RNA with spacers,

6) Example n.11 is an engineered scaffold region having a modified first region and a fourth region and a fifth region, and an engineered Cas12f1 guide RNA having a spacer,

7) Example n.12 is an engineered scaffold region having a modified second region and a fourth region and a fifth region, and an engineered Cas12f1 guide RNA having a spacer,

8) Example n.13 and Example n.14 show an engineered scaffold region having a modified first region, a second region, and a fourth region and a fifth region, and an engineered Cas12f1 guide RNA having a spacer.

In this case, n is 1, 2, or 3 for each target sequence, when n is 1, Target 1 (DY2), when n is 2, Target 2 (DY10), when n is 3, Target3 (Intergenic-22) ) is indicated.

The vectors prepared in each Example were transfected into HEK293 T cells according to Experimental Example 1.3, and indel generation efficiency was measured by Experimental Examples 1.4 to 1.5. The results of this analysis according to Experimental Example 1.6 are shown in FIGS. 2 to 13 .

2 to 4 are average indel efficiencies for Examples 1.1 to 1.13 targeting DY2, FIGS. 5 to 8 are average indel efficiencies for Examples 2.1 to 2.13 targeting DY10, FIGS. 9 to 12 are Average indel efficiency for Examples 3.1 to 3.13 targeting Intergenic-22, FIG. 13 is, Examples 1.13 to Example 1.14 targeting DY2, Examples 2.13 to Example 2.14 targeting DY10, Intergenic-22 targeting The average indel efficiencies for Examples 3.13 to 3.14 are respectively shown.

As a result of the experiment, when the engineered Cas12f1 guide RNA having an engineered scaffold region disclosed herein is used for gene editing, the Cas12f1 guide RNA having a scaffold region found in nature, and the scaffold region found in nature It can be seen that the overall gene editing efficiency is improved compared to when the tracrRNA of tracrRNA and the guide RNA in which the crRNA repeat sequence is linked with a 5'-GAAA-3' linker is used for gene editing. In addition, it can be seen that when the modifications for each region are combined (e.g.

<110> Genkore <120> An engineered guide RNA for the optimized CRISPR/Cas12f1 system and use it <130> CP21-068 <160> 441 <170> KoPatentIn 3.0 <210> 1 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> mature form of Cas12f1 tracrRNA <400> 1 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu 140 <210> 2 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> wild-type of Cas12f1 tracrRNA <400> 2 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu uuccuucca auucugcaca a 161 <210> 3 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> mature form of Cas12f1 crRNA repeat <400> 3 gaaugaagga augcaac 17 <210> 4 <211> 37 <212> RNA <213> Artificial Sequence <220> <223> wild-type of Cas12f1 crRNA repeat <400> 4 guugcagaac ccgaauagac gaaugaagga augcaac 37 <210> 5 <211> 37 <212> RNA <213> Artificial Sequence <220> <223> mature form of Cas12f1 crRNA <400> 5 gaaugaagga augcaacnnn nnnnnnnnnn nnnnnnn 37 <210> 6 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> wild-type of Cas12f1 crRNA <400> 6 guugcagaac ccgaauagac gaaugaagga augcaacnnn nnnnnnnnnn nnnnnnn 57 <210> 7 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> mature form of Cas12f1 tracrRNA + GAAA + mature form of Cas12f1 crRNA repeat <400> 7 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu gaaagaauga aggaaugcaa c 161 <210> 8 <211> 181 <212> RNA <213> Artificial Sequence <220> <223> mature form of Cas12f1 tracrRNA + GAAA + mature form of Cas12f1 crRNA <400> 8 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu gaaagaauga aggaaugcaa cnnnnnnnnn nnnnnnnnnn 180 n 181 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> 1st region of Cas12f1 guide RNA <400> 9 cuucacugau aaaguggaga a 21 <210> 10 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> 2nd region of Cas12f1 guide RNA <400> 10 ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug agugaaggug 50 <210> 11 <211> 56 <212> RNA <213> Artificial Sequence <220> <223> 3rd region of Cas12f1 guide RNA <400> 11 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucga 56 <210> 12 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> 4th region of Cas12f1 guide RNA (Mature form) <400> 12 aacaaauuca uuu 13 <210> 13 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> 4th region of Cas12f1 guide RNA (Wild-type) <400> 13 aacaaauuca uuuuuccucu ccaauucugc acaa 34 <210> 14 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> 5th region of Cas12f1 guide RNA (Mature form) <400> 14 gaaugaagga 10 <210> 15 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> 5th region of Cas12f1 guide RNA (Wild-type) <400> 15 guugcagaac ccgaauagac gaaugaagga 30 <210> 16 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> first region, 11nt deletion <400> 16 aaguggagaa 10 <210> 17 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> first region, 10nt deletion <400> 17 aaaguggaga a 11 <210> 18 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> first region, 9nt deletion <400> 18 uaaaguggag aa 12 <210> 19 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> first region, 8nt deletion <400> 19 auaaagugga gaa 13 <210> 20 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> first region, 7nt deletion <400> 20 gauaaagugg agaa 14 <210> 21 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> first region, 6nt deletion <400> 21 ugauaaagug gagaa 15 <210> 22 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> first region, 5nt deletion <400> 22 cugauaaagu ggagaa 16 <210> 23 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> first region, 4nt deletion <400> 23 acugauaaag uggagaa 17 <210> 24 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> first region, 3nt deletion <400> 24 cacugauaaa guggagaa 18 <210> 25 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> first region, 2nt deletion <400> 25 ucacugauaa aguggagaa 19 <210> 26 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> first region, 1nt deletion <400> 26 uucacugaua aaguggagaa 20 <210> 27 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 10nt <400> 27 aaaguggaga 10 <210> 28 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 11nt <400> 28 uaaaguggag a 11 <210> 29 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 12nt <400> 29 auaaagugga ga 12 <210> 30 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 13nt <400> 30 gauaaagugg aga 13 <210> 31 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 14nt <400> 31 ugauaaagug gaga 14 <210> 32 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 15nt <400> 32 cugauaaagu ggaga 15 <210> 33 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 16nt <400> 33 acugauaaag uggaga 16 <210> 34 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 17nt <400> 34 cacugauaaa guggaga 17 <210> 35 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 18nt <400> 35 ucacugauaa aguggaga 18 <210> 36 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 19nt <400> 36 uucacugaua aaguggaga 19 <210> 37 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> deleted part of the first region, 20nt <400> 37 cuucacugau aaaguggaga 20 <210> 38 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> second region, 11bp deletion <400> 38 ccgcuucacc auuagugagu gaaggugg 28 <210> 39 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> second region, 10bp deletion <400> 39 ccgcuucacc aauuaguuga gugaaggugg 30 <210> 40 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> second region, 9bp deletion <400> 40 ccgcuucacc aaauuagcuu gagugaaggu gg 32 <210> 41 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> second region, 8bp deletion <400> 41 ccgcuucacc aaaauuagac uugagugaag gugg 34 <210> 42 <211> 36 <212> RNA <213> Artificial Sequence <220> <223> second region, 7bp deletion <400> 42 ccgcuucacc aaaaguuaga acuugaguga aggugg 36 <210> 43 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> second region, 6bp deletion <400> 43 ccgcuucacc aaaagcuuag gaacuugagu gaaggugg 38 <210> 44 <211> 40 <212> RNA <213> Artificial Sequence <220> <223> second region, 5bp deletion <400> 44 ccgcuucacc aaaagcuuua gagaacuuga gugaaggugg 40 <210> 45 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> second region, 4bp deletion <400> 45 ccgcuucacc aaaagcuguu aguuagaacu ugagugaagg ugg 43 <210> 46 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> second region, 4bp+1nt deletion <400> 46 ccgcuucacc aaaagcuguu aguagaacuu gagugaaggu gg 42 <210> 47 <211> 45 <212> RNA <213> Artificial Sequence <220> <223> second region, 3bp deletion <400> 47 ccgcuucacc aaaagcuguu uagauuagaa cuugagugaa ggugg 45 <210> 48 <211> 47 <212> RNA <213> Artificial Sequence <220> <223> second region, 2bp deletion <400> 48 ccgcuucacc aaaagcuguc uuaggauuag aacuugagug aaggugg 47 <210> 49 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> second region, 1bp deletion <400> 49 ccgcuucacc aaaagcuguc cuuagggauu agaacuugag ugaaggugg 49 <210> 50 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> second region, 5' remains <400> 50 ccgcuucacc a 11 <210> 51 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> second region, 3' remains <400> 51 ugagugaagg ugg 13 <210> 52 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> upper-deleted part of the second region, 10nt <400> 52 aaagcugucc 10 <210> 53 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> upper-deleted part of the second region, 11nt <400> 53 aaagcugucc c 11 <210> 54 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> lower-deleted part of the second region, 10nt <400> 54 gauuagaacu 10 <210> 55 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> lower-deleted part of the second region, 11nt <400> 55 ggauuagaac u 11 <210> 56 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> lower-deleted part of the second region, 12nt <400> 56 gggauuagaa cu 12 <210> 57 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 10nt <400> 57 aaauuagacu 10 <210> 58 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 11nt <400> 58 aaaguuagaa cu 12 <210> 59 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 12nt <400> 59 aaagcuuagg aacu 14 <210> 60 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 13nt <400> 60 aaagcuuuag agaacu 16 <210> 61 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 14nt <400> 61 aaagcuguua guagaacu 18 <210> 62 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 15nt <400> 62 aaagcuguua guuagaacu 19 <210> 63 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 16nt <400> 63 aaagcuguuu agauuagaac u 21 <210> 64 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 17nt <400> 64 aaagcugucu uaggauuaga acu 23 <210> 65 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region, 18nt <400> 65 aaagcugucc uuagggauua gaacu 25 <210> 66 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (mature form), delete 3nt <400> 66 aacaaauuca 10 <210> 67 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (mature form), delete 4nt <400> 67 aacaaauuca u 11 <210> 68 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (mature form), delete 5nt <400> 68 aacaaauuca uu 12 <210> 69 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 21nt deletion <400> 69 aacaaauuca uuu 13 <210> 70 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 20nt deletion <400> 70 aacaaauuca uuuu 14 <210> 71 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 19nt deletion <400> 71 aacaaauuca uuuuu 15 <210> 72 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 18nt deletion <400> 72 aacaaauuca uuuuuc 16 <210> 73 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 17nt deletion <400> 73 aacaaauuca uuuuucc 17 <210> 74 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 16nt deletion <400> 74 aacaaauuca uuuuuccu 18 <210> 75 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 15nt deletion <400> 75 aacaaauuca uuuuuccuc 19 <210> 76 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 14nt deletion <400> 76 aacaaauuca uuuuuccucu 20 <210> 77 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 13nt deletion <400> 77 aacaaauuca uuuuuccucu c 21 <210> 78 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 12nt deletion <400> 78 aacaaauuca uuuuuccucu cc 22 <210> 79 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 11nt deletion <400> 79 aacaaauuca uuuuuccucu cca 23 <210> 80 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 10nt deletion <400> 80 aacaaauuca uuuuuccucu ccaa 24 <210> 81 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 9nt deletion <400> 81 aacaaauuca uuuuuccucu ccaau 25 <210> 82 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 8nt deletion <400> 82 aacaaauuca uuuuuccucu ccaauu 26 <210> 83 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 7nt deletion <400> 83 aacaaauuca uuuuuccucu ccaauuc 27 <210> 84 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 6nt deletion <400> 84 aacaaauuca uuuuuccucu ccaauucu 28 <210> 85 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 5nt deletion <400> 85 aacaaauuca uuuuuccucu ccaauucug 29 <210> 86 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 4nt deletion <400> 86 aacaaauuca uuuuuccucu ccaauucugc 30 <210> 87 <211> 31 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 3nt deletion <400> 87 aacaaauuca uuuuuccucu ccaauucugc a 31 <210> 88 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 2nt deletion <400> 88 aacaaauuca uuuuuccucu ccaauucugc ac 32 <210> 89 <211> 33 <212> RNA <213> Artificial Sequence <220> <223> fourth region of the tracrRNA (wild-type), 1nt deletion <400> 89 aacaaauuca uuuuuccucu ccaauucugc aca 33 <210> 90 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 20nt deletion <400> 90 gaaugaagga 10 <210> 91 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 19nt deletion <400> 91 cgaaugaagg a 11 <210> 92 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 18nt deletion <400> 92 acgaaugaag ga 12 <210> 93 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 17nt deletion <400> 93 gacgaaugaa gga 13 <210> 94 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 16nt deletion <400> 94 agacgaauga agga 14 <210> 95 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 15nt deletion <400> 95 uagacgaaug aagga 15 <210> 96 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 14nt deletion <400> 96 auagacgaau gaagga 16 <210> 97 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 13nt deletion <400> 97 aauagacgaa ugaagga 17 <210> 98 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 12nt deletion <400> 98 gaauagacga augaagga 18 <210> 99 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 11nt deletion <400> 99 cgaauagacg aaugaagga 19 <210> 100 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 10nt deletion <400> 100 ccgaauagac gaaugaagga 20 <210> 101 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 9nt deletion <400> 101 cccgaauaga cgaaugaagg a 21 <210> 102 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 8nt deletion <400> 102 acccgaauag acgaaugaag ga 22 <210> 103 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 7nt deletion <400> 103 aacccgaaua gacgaaugaa gga 23 <210> 104 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 6nt deletion <400> 104 gaacccgaau agacgaauga agga 24 <210> 105 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 5nt deletion <400> 105 agaacccgaa uagacgaaug aagga 25 <210> 106 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 4nt deletion <400> 106 cagaacccga auagacgaau gaagga 26 <210> 107 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 3nt deletion <400> 107 gcagaacccg aauagacgaa ugaagga 27 <210> 108 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 2nt deletion <400> 108 ugcagaaccc gaauagacga augaagga 28 <210> 109 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> fifth region of the crRNA (wild-type), 1nt deletion <400> 109 uugcagaacc cgaauagacg aaugaagga 29 <210> 110 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 7bp deletion (mature form) <400> 110 aacaaagaaa gga 13 <210> 111 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 6bp deletion (mature form) <400> 111 aacaaaugaa aagga 15 <210> 112 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 5bp deletion (mature form) <400> 112 aacaaauuga aaaagga 17 <210> 113 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 4bp deletion (mature form) <400> 113 aacaaauucg aaagaagga 19 <210> 114 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 3bp deletion (mature form) <400> 114 aacaaauuca gaaaugaagg a 21 <210> 115 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 2bp deletion (mature form) <400> 115 aacaaauuca ugaaaaugaa gga 23 <210> 116 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region, 1bp deletion (mature form) <400> 116 aacaaauuca uugaaaaaug aagga 25 <210> 117 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> fourth region + Linker + fifth region (mature form) <400> 117 aacaaauuca uuugaaagaa ugaagga 27 <210> 118 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 20nt deletion in the first region) <400> 118 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 120 <210> 119 <211> 121 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 19nt deletion in the first region) <400> 119 aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg ugggcugcuu 60 gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa caaauucauu 120 u 121 <210> 120 <211> 122 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 18nt deletion in the first region) <400> 120 gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag gugggcugcu 60 ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa acaaauucau 120 uu 122 <210> 121 <211> 123 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 17nt deletion in the first region) <400> 121 agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa ggugggcugc 60 uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga aacaaauuca 120 uuu 123 <210> 122 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 16nt deletion in the first region) <400> 122 gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga aggugggcug 60 cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg aaacaaauuc 120 auuu 124 <210> 123 <211> 125 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 15nt deletion in the first region) <400> 123 ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuu 125 <210> 124 <211> 126 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 14nt deletion in the first region) <400> 124 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuu 126 <210> 125 <211> 127 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 13nt deletion in the first region) <400> 125 guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 127 <210> 126 <211> 128 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 12nt deletion in the first region) <400> 126 aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga gugaaggugg 60 gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc cucgaaacaa 120 128 <210> 127 <211> 129 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 11nt deletion in the first region) <400> 127 aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuu 129 <210> 128 <211> 130 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 10nt deletion in the first region) <400> 128 aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu 60 gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac 120 aaauucauuu 130 <210> 129 <211> 131 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 9nt deletion in the first region) <400> 129 uaaaguggag aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu u 131 <210> 130 <211> 132 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 8nt deletion in the first region) <400> 130 auaaagugga gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 caaauucau uu 132 <210> 131 <211> 133 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 7nt deletion in the first region) <400> 131 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuu 133 <210> 132 <211> 134 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 6nt deletion in the first region) <400> 132 ugauaaagug gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuu 134 <210> 133 <211> 135 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 5nt deletion in the first region) <400> 133 cugauaaagu ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug 60 aaggugggcu gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc 120 gaaacaaauu cauuu 135 <210> 134 <211> 136 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 4nt deletion in the first region) <400> 134 acugauaaag uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuu 136 <210> 135 <211> 137 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 3nt deletion in the first region) <400> 135 cacugauaaa guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag 60 ugaagguggg cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc 120 ucgaaacaaa uucauuu 137 <210> 136 <211> 138 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 2nt deletion in the first region) <400> 136 ucacugauaa aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuu 138 <210> 137 <211> 139 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 1nt deletion in the first region) <400> 137 uucacugaua aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug 60 agugaaggug ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac 120 ccucgaaaca aauucauuu 139 <210> 138 <211> 117 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 10bp deletion in the second) region) <400> 138 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa uucauuu 117 <210> 139 <211> 119 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 9bp deletion in the second) region) <400> 139 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuu 119 <210> 140 <211> 122 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 8bp deletion in the second) region) <400> 140 cuucacugau aaaguggaga accgcuucac caaauuagac uugagugaag gugggcugcu 60 ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa acaaauucau 120 uu 122 <210> 141 <211> 125 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 7bp deletion in the second) region) <400> 141 cuucacugau aaaguggaga accgcuucac caaaaguuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuu 125 <210> 142 <211> 127 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 6bp deletion in the second) region) <400> 142 cuucacugau aaaguggaga accgcuucac caaaagcuua ggaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 127 <210> 143 <211> 129 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 5bp deletion in the second) region) <400> 143 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuu 129 <210> 144 <211> 132 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 4bp deletion in the second) region) <400> 144 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 caaauucau uu 132 <210> 145 <211> 131 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 4bp+1nt deletion in the second) region) <400> 145 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu u 131 <210> 146 <211> 134 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 3bp deletion in the second) region) <400> 146 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuu 134 <210> 147 <211> 136 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 2bp deletion in the second) region) <400> 147 cuucacugau aaaguggaga accgcuucac caaaagcugu cuuaggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuu 136 <210> 148 <211> 138 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 1bp deletion in the second) region) <400> 148 cuucacugau aaaguggaga accgcuucac caaaagcugu ccuuagggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuu 138 <210> 149 <211> 133 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 7nt deletion in the fourth region) <400> 149 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaa 133 <210> 150 <211> 134 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 6nt deletion in the fourth region) <400> 150 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaau 134 <210> 151 <211> 135 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 5nt deletion in the fourth region) <400> 151 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauu 135 <210> 152 <211> 136 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 4nt deletion in the fourth region) <400> 152 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauuc 136 <210> 153 <211> 137 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 3nt deletion in the fourth region) <400> 153 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauuca 137 <210> 154 <211> 138 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 2nt deletion in the fourth region) <400> 154 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucau 138 <210> 155 <211> 139 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, 1nt deletion in the fourth region) <400> 155 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauu 139 <210> 156 <211> 97 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, modified first region and modified second region) <400> 156 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa uucauuu 97 <210> 157 <211> 113 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, modified first region and modified fourth and fifth region) <400> 157 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaa 113 <210> 158 <211> 110 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, modified second region and modified fourth and fifth region) <400> 158 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 110 <210> 159 <211> 90 <212> RNA <213> Artificial Sequence <220> <223> engineered tracrRNA (mature form, modified first region, modified second region, modified fourth and fifth region) <400> 159 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa 90 <210> 160 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 7nt deletion in the fifth region) <400> 160 ggaaugcaac 10 <210> 161 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 6nt deletion in the fifth region) <400> 161 aggaaugcaa c 11 <210> 162 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 5nt deletion in the fifth region) <400> 162 aaggaaugca ac 12 <210> 163 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 4nt deletion in the fifth region) <400> 163 gaaggaaugc aac 13 <210> 164 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 3nt deletion in the fifth region) <400> 164 ugaaggaaug caac 14 <210> 165 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 2nt deletion in the fifth region) <400> 165 augaaggaau gcaac 15 <210> 166 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> engineered crRNA repeat (mature form, 1nt deletion in the fifth region) <400> 166 aaugaaggaa ugcaac 16 <210> 167 <211> 141 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 20nt deletion in the first region) <400> 167 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 gaaagaauga aggaaugcaa c 141 <210> 168 <211> 142 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 19nt deletion in the first region) <400> 168 aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg ugggcugcuu 60 gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa caaauucauu 120 ugaaagaaug aaggaaugca ac 142 <210> 169 <211> 143 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 18nt deletion in the first region) <400> 169 gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag gugggcugcu 60 ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa acaaauucau 120 uugaaagaau gaaggaaugc aac 143 <210> 170 <211> 144 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 17nt deletion in the first region) <400> 170 agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa ggugggcugc 60 uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga aacaaauuca 120 uuugaaagaa ugaaggaaug caac 144 <210> 171 <211> 145 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 16nt deletion in the first region) <400> 171 gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga aggugggcug 60 cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg aaacaaauuc 120 auuugaaaga augaaggaau gcaac 145 <210> 172 <211> 146 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 15nt deletion in the first region) <400> 172 ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuugaaag aaugaaggaa ugcaac 146 <210> 173 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 14nt deletion in the first region) <400> 173 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuugaaa gaaugaagga augcaac 147 <210> 174 <211> 148 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 13nt deletion in the first region) <400> 174 guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 uucauuugaa agaaugaagg aaugcaac 148 <210> 175 <211> 149 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 12nt deletion in the first region) <400> 175 aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga gugaaggugg 60 gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc cucgaaacaa 120 auucauuuga aagaaugaag gaaugcaac 149 <210> 176 <211> 150 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 11nt deletion in the first region) <400> 176 aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac 150 <210> 177 <211> 151 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 10nt deletion in the first region) <400> 177 aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu 60 gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac 120 aaauucauuu gaaagaauga aggaaugcaa c 151 <210> 178 <211> 152 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 9nt deletion in the first region) <400> 178 uaaaguggag aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu ugaaagaaug aaggaaugca ac 152 <210> 179 <211> 153 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 8nt deletion in the first region) <400> 179 auaaagugga gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 acaaauucau uugaaagaau gaaggaaugc aac 153 <210> 180 <211> 154 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 7nt deletion in the first region) <400> 180 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuugaaagaa ugaaggaaug caac 154 <210> 181 <211> 155 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 6nt deletion in the first region) <400> 181 ugauaaagug gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaac 155 <210> 182 <211> 156 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 5nt deletion in the first region) <400> 182 cugauaaagu ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug 60 aaggugggcu gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc 120 gaaacaaauu cauuugaaag aaugaaggaa ugcaac 156 <210> 183 <211> 157 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 4nt deletion in the first region) <400> 183 acugauaaag uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuugaaa gaaugaagga augcaac 157 <210> 184 <211> 158 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 3nt deletion in the first region) <400> 184 cacugauaaa guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag 60 ugaagguggg cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc 120 ucgaaacaaa uucauuugaa agaaugaagg aaugcaac 158 <210> 185 <211> 159 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 2nt deletion in the first region) <400> 185 ucacugauaa aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuuga aagaaugaag gaaugcaac 159 <210> 186 <211> 160 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 1nt deletion in the first region) <400> 186 uucacugaua aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug 60 agugaaggug ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac 120 ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac 160 <210> 187 <211> 138 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 11bp deletion in the second) region) <400> 187 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa uucauuugaa 120 agaaugaagg aaugcaac 138 <210> 188 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 10bp deletion in the second region) <400> 188 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug 120 aaagaaugaa ggaaugcaac 140 <210> 189 <211> 142 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 9bp deletion in the second) region) <400> 189 cuucacugau aaaguggaga accgcuucac caaauuagcu ugagugaagg ugggcugcuu 60 gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa caaauucauu 120 ugaaagaaug aaggaaugca ac 142 <210> 190 <211> 144 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 8bp deletion in the second region) <400> 190 cuucacugau aaaguggaga accgcuucac caaaauuaga cuugagugaa ggugggcugc 60 uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga aacaaauuca 120 uuugaaagaa ugaaggaaug caac 144 <210> 191 <211> 146 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 7bp deletion in the second) region) <400> 191 cuucacugau aaaguggaga accgcuucac caaaaguuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuugaaag aaugaaggaa ugcaac 146 <210> 192 <211> 148 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 6bp deletion in the second) region) <400> 192 cuucacugau aaaguggaga accgcuucac caaaagcuua ggaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 uucauuugaa agaaugaagg aaugcaac 148 <210> 193 <211> 150 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 5bp deletion in the second region) <400> 193 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac 150 <210> 194 <211> 152 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 4bp deletion in the second region) <400> 194 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu ugaaagaaug aaggaaugca ac 152 <210> 195 <211> 153 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 4bp+1nt deletion in the second region) <400> 195 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 acaaauucau uugaaagaau gaaggaaugc aac 153 <210> 196 <211> 155 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 3bp deletion in the second) region) <400> 196 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaac 155 <210> 197 <211> 157 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 2bp deletion in the second) region) <400> 197 cuucacugau aaaguggaga accgcuucac caaaagcugu cuuaggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuugaaa gaaugaagga augcaac 157 <210> 198 <211> 159 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold(mature form, 1bp deletion in the second region) <400> 198 cuucacugau aaaguggaga accgcuucac caaaagcugu ccuuagggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuuga aagaaugaag gaaugcaac 159 <210> 199 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 7bp deletion in the fourth and fifth region) <400> 199 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaagaaagga augcaac 147 <210> 200 <211> 149 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 6bp deletion in the fourth and fifth region) <400> 200 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaaugaaaag gaaugcaac 149 <210> 201 <211> 151 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 5bp deletion in the fourth and fifth region) <400> 201 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauugaaaa aggaaugcaa c 151 <210> 202 <211> 153 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 4bp deletion in the fourth and fifth region) <400> 202 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgaaa gaaggaaugc aac 153 <210> 203 <211> 155 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 3bp deletion in the fourth and fifth region) <400> 203 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucagaa augaaggaau gcaac 155 <210> 204 <211> 157 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 2bp deletion in the fourth and fifth region) <400> 204 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauga aaaugaagga augcaac 157 <210> 205 <211> 159 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, 1bp deletion in the fourth and fifth region) <400> 205 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauug aaaaaugaag gaaugcaac 159 <210> 206 <211> 118 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, modified first and second region) <400> 206 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa uucauuugaa agaaugaagg aaugcaac 118 <210> 207 <211> 127 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, modified first, fourth and fifth region) <400> 207 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaac 127 <210> 208 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, modified second, fourth and fifth region) <400> 208 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug 120 caac 124 <210> 209 <211> 104 <212> RNA <213> Artificial Sequence <220> <223> engineered scaffold (mature form, modified first, second, fourth and fifth region) <400> 209 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caac 104 <210> 210 <211> 189 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 1nt deletion in the first region) <400> 210 uucacugaua aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug 60 agugaaggug ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac 120 ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac nnnnnnnnnn nnnnnnnnnn 180 189 <210> 211 <211> 188 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 2nt deletion in the first region) <400> 211 ucacugauaa aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuuga aagaaugaag gaaugcaacn nnnnnnnnnn nnnnnnnnnu 180 188 <210> 212 <211> 187 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 3nt deletion in the first region) <400> 212 cacugauaaa guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag 60 ugaagguggg cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc 120 ucgaaacaaa uucauuugaa agaaugaagg aaugcaacnn nnnnnnnnnn nnnnnnnnuu 180 187 <210> 213 <211> 186 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 4nt deletion in the first region) <400> 213 acugauaaag uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuugaaa gaaugaagga augcaacnnn nnnnnnnnnn nnnnnnnuuu 180 186 <210> 214 <211> 185 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 5nt deletion in the first region) <400> 214 cugauaaagu ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug 60 aaggugggcu gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc 120 gaaacaaauu cauuugaaag aaugaaggaa ugcaacnnnn nnnnnnnnnn nnnnnnuuuu 180 185 <210> 215 <211> 184 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 6nt deletion in the first region) <400> 215 ugauaaagug gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaacnnnnn nnnnnnnnnn nnnnnuuuua 180 uuuu 184 <210> 216 <211> 183 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 7nt deletion in the first region) <400> 216 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuugaaagaa ugaaggaaug caacnnnnnn nnnnnnnnnn nnnnuuuuau 180 uuu 183 <210> 217 <211> 182 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 8nt deletion in the first region) <400> 217 auaaagugga gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 acaaauucau uugaaagaau gaaggaaugc aacnnnnnnn nnnnnnnnnn nnnuuuuauu 180 uu 182 <210> 218 <211> 181 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 9nt deletion in the first region) <400> 218 uaaaguggag aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu ugaaagaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nnuuuuauuu 180 u 181 <210> 219 <211> 180 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 10nt deletion in the first region) <400> 219 aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu 60 gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac 120 aaauucauuu gaaagaauga aggaaugcaa cnnnnnnnnn nnnnnnnnnn nuuuuauuuu 180 180 <210> 220 <211> 179 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 11nt deletion in the first region) <400> 220 aaguggagaa ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac nnnnnnnnnn nnnnnnnnnn uuuuauuuu 179 <210> 221 <211> 170 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 12nt deletion in the first region) <400> 221 aguggagaac cgcuucacca aaagcugucc cuuaggggau uagaacuuga gugaaggugg 60 gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc cucgaaacaa 120 auucauuuga aagaaugaag gaaugcaacn nnnnnnnnnn nnnnnnnnnu 170 <210> 222 <211> 168 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 13nt deletion in the first region) <400> 222 guggagaacc gcuucaccaa aagcuguccc uuaggggauu agaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 uucauuugaa agaaugaagg aaugcaacnn nnnnnnnnnn nnnnnnnn 168 <210> 223 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 14nt deletion in the first region) <400> 223 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuugaaa gaaugaagga augcaacnnn nnnnnnnnnn nnnnnnn 167 <210> 224 <211> 166 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 15nt deletion in the first region) <400> 224 ggagaaccgc uucaccaaaa gcugucccuu aggggauuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuugaaag aaugaaggaa ugcaacnnnn nnnnnnnnnn nnnnnn 166 <210> 225 <211> 165 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 16nt deletion in the first region) <400> 225 gagaaccgcu ucaccaaaag cugucccuua ggggauuaga acuugaguga aggugggcug 60 cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg aaacaaauuc 120 auuugaaaga augaaggaau gcaacnnnnn nnnnnnnnnn nnnnn 165 <210> 226 <211> 164 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 17nt deletion in the first region) <400> 226 agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa ggugggcugc 60 uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga aacaaauuca 120 uuugaaagaa ugaaggaaug caacnnnnnn nnnnnnnnnn nnnn 164 <210> 227 <211> 163 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 18nt deletion in the first region) <400> 227 gaaccgcuuc accaaaagcu gucccuuagg ggauuagaac uugagugaag gugggcugcu 60 ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa acaaauucau 120 uugaaagaau gaaggaaugc aacnnnnnnn nnnnnnnnnn nnn 163 <210> 228 <211> 162 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 19nt deletion in the first region) <400> 228 aaccgcuuca ccaaaagcug ucccuuaggg gauuagaacu ugagugaagg ugggcugcuu 60 gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa caaauucauu 120 ugaaagaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nn 162 <210> 229 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 20nt deletion in the first region) <400> 229 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 gaaagaauga aggaaugcaa cnnnnnnnnn nnnnnnnnnn n 161 <210> 230 <211> 179 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 1bp deletion in the second region) <400> 230 cuucacugau aaaguggaga accgcuucac caaaagcugu ccuuagggau uagaacuuga 60 gugaaggugg gcugcuugca ucagccuaau gucgagaagu gcuuucuucg gaaaguaacc 120 cucgaaacaa auucauuuga aagaaugaag gaaugcaacn nnnnnnnnnn nnnnnnnnnn 179 <210> 231 <211> 177 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 2bp deletion in the second region) <400> 231 cuucacugau aaaguggaga accgcuucac caaaagcugu cuuaggauua gaacuugagu 60 gaaggugggc ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu 120 cgaaacaaau ucauuugaaa gaaugaagga augcaacnnn nnnnnnnnnn nnnnnnn 177 <210> 232 <211> 175 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 3bp deletion in the second region) <400> 232 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaacnnnnn nnnnnnnnnn nnnnn 175 <210> 233 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 4bp+1nt deletion in the second region) <400> 233 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguuagaac uugagugaag 60 gugggcugcu ugcaucagcc uaaugucgag aagugcuuuc uucggaaagu aacccucgaa 120 acaaauucau uugaaagaau gaaggaaugc aacnnnnnnn nnnnnnnnnn nnn 173 <210> 234 <211> 172 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 4bp deletion in the second region) <400> 234 cuucacugau aaaguggaga accgcuucac caaaagcugu uaguagaacu ugagugaagg 60 ugggcugcuu gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa 120 caaauucauu ugaaagaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nn 172 <210> 235 <211> 170 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 5bp deletion in the second region) <400> 235 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac nnnnnnnnnn nnnnnnnnnn 170 <210> 236 <211> 168 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 6bp deletion in the second region) <400> 236 cuucacugau aaaguggaga accgcuucac caaaagcuua ggaacuugag ugaagguggg 60 cugcuugcau cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa 120 uucauuugaa agaaugaagg aaugcaacnn nnnnnnnnnn nnnnnnnn 168 <210> 237 <211> 166 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 7bp deletion in the second region) <400> 237 cuucacugau aaaguggaga accgcuucac caaaaguuag aacuugagug aaggugggcu 60 gcuugcauca gccuaauguc gagaagugcu uucuucggaa aguaacccuc gaaacaaauu 120 cauuugaaag aaugaaggaa ugcaacnnnn nnnnnnnnnn nnnnnn 166 <210> 238 <211> 164 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 8bp deletion in the second region) <400> 238 cuucacugau aaaguggaga accgcuucac caaaauuaga cuugagugaa ggugggcugc 60 uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga aacaaauuca 120 uuugaaagaa ugaaggaaug caacnnnnnn nnnnnnnnnn nnnn 164 <210> 239 <211> 162 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 9bp deletion in the second region) <400> 239 cuucacugau aaaguggaga accgcuucac caaauuagcu ugagugaagg ugggcugcuu 60 gcaucagccu aaugucgaga agugcuuucu ucggaaagua acccucgaaa caaauucauu 120 ugaaagaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nn 162 <210> 240 <211> 160 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 10bp deletion in the second region) <400> 240 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug 120 aaagaaugaa ggaaugcaac nnnnnnnnnn nnnnnnnnnn 160 <210> 241 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 11bp deletion in the second region) <400> 241 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa uucauuugaa 120 agaaugaagg aaugcaacnn nnnnnnnnnn nnnnnnnnuu uuauuuu 167 <210> 242 <211> 179 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 1bp deletion in the fourth and fifth region) <400> 242 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauug aaaaaugaag gaaugcaacn nnnnnnnnnn nnnnnnnnn 179 <210> 243 <211> 177 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 2bp deletion in the fourth and fifth region) <400> 243 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauga aaaugaagga augcaacnnn nnnnnnnnnn nnnnnnn 177 <210> 244 <211> 175 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 3bp deletion in the fourth and fifth region) <400> 244 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucagaa augaaggaau gcaacnnnnn nnnnnnnnnn nnnnn 175 <210> 245 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 4bp deletion in the fourth and fifth region) <400> 245 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgaaa gaaggaaugc aacnnnnnnn nnnnnnnnnn nnn 173 <210> 246 <211> 171 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 5bp deletion in the fourth and fifth region) <400> 246 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauugaaaa aggaaugcaa cnnnnnnnnn nnnnnnnnnn n 171 <210> 247 <211> 169 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 6bp deletion in the fourth and fifth region) <400> 247 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaaugaaaag gaaugcaacn nnnnnnnnnn nnnnnnnnn 169 <210> 248 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, 7bp deletion in the fourth and fifth region) <400> 248 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaagaaagga augcaacnnn nnnnnnnnnn nnnnnnn 167 <210> 249 <211> 138 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first and second region) <400> 249 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa uucauuugaa agaaugaagg aaugcaacnn 120 nnnnnnnnnn nnnnnnnn 138 <210> 250 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, fourth and fifth region) <400> 250 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaacnnn nnnnnnnnnn nnnnnnn 147 <210> 251 <211> 144 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified second, fourth and fifth region) <400> 251 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug 120 caacnnnnnn nnnnnnnnnn nnnn 144 <210> 252 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 252 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 253 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 253 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 254 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 254 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 255 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 255 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 256 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 256 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 257 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 257 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 258 <211> 124 <212> RNA <213> Artificial Sequence <220> <223> sgRNA (mature form, modified first, second, fourth and fifth region) <400> 258 accgcuucac cauuagugag ugaagguggg cugcuugcau cagccuaaug ucgagaagug 60 cuuucuucgg aaaguaaccc ucgaaacaaa gaaaggaaug caacnnnnnn nnnnnnnnnn 120 nnnn 124 <210> 259 <211> 529 <212> PRT <213> Artificial Sequence <220> <223> Cas14a1 amino acid sequence <400> 259 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 260 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> N-terminal NLS + Cas14a1 amino acid sequence <400> 260 Met Pro Lys Lys Lys Arg Lys Val Ala Lys Asn Thr Ile Thr Lys Thr 1 5 10 15 Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu Val Glu Lys 20 25 30 Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala Leu Glu Lys 35 40 45 Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu Lys Val Ala 50 55 60 Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu Phe Cys Lys 65 70 75 80 Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg Gly Gln Phe 85 90 95 Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe Arg Gln Leu 100 105 110 Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile Glu Leu Tyr 115 120 125 Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala Ser Ser Val 130 135 140 Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala Glu Leu Phe 145 150 155 160 Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile Lys Ser Asn 165 170 175 Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu Pro Thr Thr 180 185 190 Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys Gly Gly Gln 195 200 205 Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe Ile Ile Lys 210 215 220 Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp Lys Tyr Arg 225 230 235 240 Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser Pro Lys Pro 245 250 255 Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn Lys Gly Trp 260 265 270 Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val Met Asn Gly 275 280 285 Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser Lys Ile Gly 290 295 300 Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val Pro Lys Ile 305 310 315 320 Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp Val Gly Val 325 330 335 Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser Arg Tyr Ser 340 345 350 Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met Phe Ala Arg 355 360 365 Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala Gly His Gly 370 375 380 Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu Lys Ser Glu 385 390 395 400 Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu Ile Ala Asp 405 410 415 Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu Asn Leu Glu 420 425 430 Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg Leu Arg Gly 435 440 445 Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe Lys Leu Lys 450 455 460 Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn Thr Ser Lys 465 470 475 480 Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn Phe Glu Tyr 485 490 495 Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys Cys Asn Phe 500 505 510 Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser Asn Pro Lys 515 520 525 Leu Lys Ser Thr Lys Glu Glu Pro 530 535 <210> 261 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> C-terminal NLS + Cas14a1 amino acid sequence <400> 261 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro Pro Lys Lys Lys Arg Lys Val 530 535 <210> 262 <211> 543 <212> PRT <213> Artificial Sequence <220> <223> N/C-terminal NLS + Cas14a1 amino acid sequence <400> 262 Met Pro Lys Lys Lys Arg Lys Val Ala Lys Asn Thr Ile Thr Lys Thr 1 5 10 15 Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu Val Glu Lys 20 25 30 Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala Leu Glu Lys 35 40 45 Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu Lys Val Ala 50 55 60 Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu Phe Cys Lys 65 70 75 80 Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg Gly Gln Phe 85 90 95 Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe Arg Gln Leu 100 105 110 Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile Glu Leu Tyr 115 120 125 Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala Ser Ser Val 130 135 140 Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala Glu Leu Phe 145 150 155 160 Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile Lys Ser Asn 165 170 175 Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu Pro Thr Thr 180 185 190 Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys Gly Gly Gln 195 200 205 Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe Ile Ile Lys 210 215 220 Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp Lys Tyr Arg 225 230 235 240 Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser Pro Lys Pro 245 250 255 Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn Lys Gly Trp 260 265 270 Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val Met Asn Gly 275 280 285 Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser Lys Ile Gly 290 295 300 Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val Pro Lys Ile 305 310 315 320 Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp Val Gly Val 325 330 335 Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser Arg Tyr Ser 340 345 350 Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met Phe Ala Arg 355 360 365 Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala Gly His Gly 370 375 380 Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu Lys Ser Glu 385 390 395 400 Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu Ile Ala Asp 405 410 415 Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu Asn Leu Glu 420 425 430 Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg Leu Arg Gly 435 440 445 Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe Lys Leu Lys 450 455 460 Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn Thr Ser Lys 465 470 475 480 Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn Phe Glu Tyr 485 490 495 Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys Cys Asn Phe 500 505 510 Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser Asn Pro Lys 515 520 525 Leu Lys Ser Thr Lys Glu Glu Pro Pro Lys Lys Lys Arg Lys Val 530 535 540 <210> 263 <211> 1003 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence (N terminal -Cytidine deaminase) <400> 263 Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala 1 5 10 15 Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val 20 25 30 Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu 35 40 45 Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn 50 55 60 Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu 65 70 75 80 Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser 85 90 95 Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser 100 105 110 Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His 115 120 125 His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser 130 135 140 Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp 145 150 155 160 Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg 165 170 175 Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile 180 185 190 Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro 195 200 205 Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg 210 215 220 Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser 225 230 235 240 Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala 245 250 255 Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ala Lys Asn Thr 260 265 270 Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala 275 280 285 Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile 290 295 300 Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His 305 310 315 320 Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys 325 330 335 Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu 340 345 350 Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile 355 360 365 Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu 370 375 380 Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn 385 390 395 400 Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala 405 410 415 Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys 420 425 430 Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly 435 440 445 Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln 450 455 460 Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp 465 470 475 480 Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile 485 490 495 Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys 500 505 510 Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg 515 520 525 Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys 530 535 540 Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly 545 550 555 560 Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp 565 570 575 Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile 580 585 590 Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe 595 600 605 Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys 610 615 620 Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg 625 630 635 640 Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr 645 650 655 Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys 660 665 670 Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met 675 680 685 Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile 690 695 700 Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu 705 710 715 720 Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn 725 730 735 Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe 740 745 750 Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu 755 760 765 Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile 770 775 780 Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro Ser Gly Gly Ser 785 790 795 800 Gly Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu 805 810 815 Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu 820 825 830 Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val 835 840 845 His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr 850 855 860 Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser 865 870 875 880 Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly Ser 885 890 895 Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys 900 905 910 Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu 915 920 925 Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala 930 935 940 Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala 945 950 955 960 Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu 965 970 975 Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly 980 985 990 Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val 995 1000 <210> 264 <211> 1003 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence (C terminal -Cytidine deaminase) <400> 264 Met Pro Lys Lys Lys Arg Lys Val Ala Lys Asn Thr Ile Thr Lys Thr 1 5 10 15 Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu Val Glu Lys 20 25 30 Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala Leu Glu Lys 35 40 45 Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu Lys Val Ala 50 55 60 Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu Phe Cys Lys 65 70 75 80 Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg Gly Gln Phe 85 90 95 Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe Arg Gln Leu 100 105 110 Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile Glu Leu Tyr 115 120 125 Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala Ser Ser Val 130 135 140 Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala Glu Leu Phe 145 150 155 160 Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile Lys Ser Asn 165 170 175 Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu Pro Thr Thr 180 185 190 Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys Gly Gly Gln 195 200 205 Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe Ile Ile Lys 210 215 220 Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp Lys Tyr Arg 225 230 235 240 Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser Pro Lys Pro 245 250 255 Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn Lys Gly Trp 260 265 270 Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val Met Asn Gly 275 280 285 Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser Lys Ile Gly 290 295 300 Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val Pro Lys Ile 305 310 315 320 Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp Val Gly Val 325 330 335 Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser Arg Tyr Ser 340 345 350 Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met Phe Ala Arg 355 360 365 Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala Gly His Gly 370 375 380 Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu Lys Ser Glu 385 390 395 400 Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu Ile Ala Asp 405 410 415 Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu Asn Leu Glu 420 425 430 Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg Leu Arg Gly 435 440 445 Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe Lys Leu Lys 450 455 460 Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn Thr Ser Lys 465 470 475 480 Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn Phe Glu Tyr 485 490 495 Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys Cys Asn Phe 500 505 510 Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser Asn Pro Lys 515 520 525 Leu Lys Ser Thr Lys Glu Glu Pro Ser Gly Gly Ser Ser Gly Gly Ser 530 535 540 Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 545 550 555 560 Ser Gly Gly Ser Ser Gly Gly Ser Ser Ser Glu Thr Gly Pro Val Ala 565 570 575 Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val 580 585 590 Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu 595 600 605 Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn 610 615 620 Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu 625 630 635 640 Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser 645 650 655 Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser 660 665 670 Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His 675 680 685 His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser 690 695 700 Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp 705 710 715 720 Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg 725 730 735 Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile 740 745 750 Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro 755 760 765 Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg 770 775 780 Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser 785 790 795 800 Gly Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu 805 810 815 Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu 820 825 830 Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val 835 840 845 His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr 850 855 860 Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser 865 870 875 880 Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly Ser 885 890 895 Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys 900 905 910 Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu 915 920 925 Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala 930 935 940 Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala 945 950 955 960 Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu 965 970 975 Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly 980 985 990 Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val 995 1000 <210> 265 <211> 941 <212> PRT <213> Artificial Sequence <220> <223> DNA sequence (N terminal -Adenine deaminase) <400> 265 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser 165 170 175 Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser 180 185 190 Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr 195 200 205 Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg 210 215 220 Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly 225 230 235 240 Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala 245 250 255 Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg 260 265 270 Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys 275 280 285 Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val 290 295 300 Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His 305 310 315 320 Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala 325 330 335 Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln 340 345 350 Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly 355 360 365 Ser Ser Gly Gly Ser Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 370 375 380 Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ala Lys Asn 385 390 395 400 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 405 410 415 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 420 425 430 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 435 440 445 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 450 455 460 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 465 470 475 480 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 485 490 495 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 500 505 510 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 515 520 525 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 530 535 540 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 545 550 555 560 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 565 570 575 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 580 585 590 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 595 600 605 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 610 615 620 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 625 630 635 640 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 645 650 655 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 660 665 670 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 675 680 685 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 690 695 700 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 705 710 715 720 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 725 730 735 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 740 745 750 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 755 760 765 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 770 775 780 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 785 790 795 800 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 805 810 815 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 820 825 830 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 835 840 845 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 850 855 860 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 865 870 875 880 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 885 890 895 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 900 905 910 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro Lys Arg Pro 915 920 925 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 930 935 940 <210> 266 <211> 941 <212> PRT <213> Artificial Sequence <220> <223> DNA sequence (C terminal -Adenine deaminase) <400> 266 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly 530 535 540 Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly 545 550 555 560 Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 565 570 575 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 580 585 590 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 595 600 605 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 610 615 620 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 625 630 635 640 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 645 650 655 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 660 665 670 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 675 680 685 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 690 695 700 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 705 710 715 720 Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser 725 730 735 Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser 740 745 750 Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr 755 760 765 Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg 770 775 780 Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly 785 790 795 800 Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala 805 810 815 Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg 820 825 830 Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys 835 840 845 Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val 850 855 860 Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His 865 870 875 880 Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala 885 890 895 Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln 900 905 910 Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Lys Arg Pro 915 920 925 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 930 935 940 <210> 267 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> Human codon-optimized Cas14a1 with NLS <400> 267 atgccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccgc caagaacaca 60 attacaaaga cactgaagct gaggatcgtg agaccataca acagcgctga ggtcgagaag 120 attgtggctg atgaaaagaa caacagggaa aagatcgccc tcgagaagaa caaggataag 180 gtgaaggagg cctgctctaa gcacctgaaa gtggccgcct actgcaccac acaggtggag 240 aggaacgcct gtctgttttg taaagctcgg aagctggatg ataagtttta ccagaagctg 300 cggggccagt tccccgatgc cgtcttttgg caggagatta gcgagatctt cagacagctg 360 cagaagcagg ccgccgagat ctacaaccag agcctgatcg agctctacta cgagatcttc 420 atcaagggca agggcattgc caacgcctcc tccgtggagc actacctgag cgacgtgtgc 480 tacacaagag ccgccgagct ctttaagaac gccgctatcg cttccgggct gaggagcaag 540 attaagagta acttccggct caaggagctg aagaacatga agagcggcct gcccactaca 600 aagagcgaca acttcccaat tccactggtg aagcagaagg ggggccagta cacagggttc 660 gagattcca accacaacag cgactttatt attaagatcc cctttggcag gtggcaggtc 720 aagaaggaga ttgacaagta caggccctgg gagaagtttg atttcgagca ggtgcagaag 780 agccccaagc ctatttccct gctgctgtcc acacagcggc ggaagaggaa caaggggtgg 840 tctaaggatg aggggaccga ggccgagatt aagaaagtga tgaacggcga ctaccagaca 900 agctacatcg aggtcaagcg gggcagtaag attggcgaga agagcgcctg gatgctgaac 960 ctgagcattg acgtgccaaa gattgataag ggcgtggatc ccagcatcat cggagggatc 1020 gatgtggggg tcaagagccc cctcgtgtgc gccatcaaca acgccttcag caggtacagc 1080 atctccgata acgacctgtt ccactttaac aagaagatgt tcgcccggcg gaggattttg 1140 ctcaagaaga accggcacaa gcgggccgga cacggggcca agaacaagct caagcccatc 1200 actatcctga ccgagaagag cgagaggttc aggaagaagc tcatcgagag atgggcctgc 1260 gagatcgccg atttctttat taagaacaag gtcggaacag tgcagatgga gaacctcgag 1320 agcatgaaga ggaaggagga ttcctacttc aacattcggc tgagggggtt ctggccctac 1380 gctgagatgc agaacaagat tgagtttaag ctgaagcagt acgggattga gatccggaag 1440 gtggccccca acaacaccag caagacctgc agcaagtgcg ggcacctcaa caactacttc 1500 aacttcgagt accggaagaa gaacaagttc ccacacttca agtgcgagaa gtgcaacttt 1560 aaggagaacg ccgattacaa cgccgccctg aacatcagca accctaagct gaagagcact 1620 aaggaggagc ccaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 1680 1680 <210> 268 <211> 3009 <212> DNA <213> Artificial Sequence <220> <223> Deaminase fusion Cas14a1 <400> 268 atgccaaaga agaagcggaa agtctcctca gagactgggc ctgtcgccgt cgatccaacc 60 ctgcgccgcc ggattgaacc tcacgagttt gaagtgttct ttgacccccg ggagctgaga 120 aaggagacat gcctgctgta cgagatcaac tggggaggca ggcactccat ctggaggcac 180 acctctcaga acacaaataa gcacgtggag gtgaacttca tcgagaagtt taccacagag 240 cggtacttct gccccaatac cagatgtagc atcacatggt ttctgagctg gtccccttgc 300 ggagagtgta gcagggccat caccgagttc ctgtccagat atccacacgt gacactgttt 360 atctacatcg ccaggctgta tcaccacgca gacccaagga ataggcaggg cctgcgcgat 420 ctgatcagct ccggcgtgac catccagatc atgacagagc aggagtccgg ctactgctgg 480 cggaacttcg tgaattattc tcctagcaac gaggcccact ggcctaggta cccacacctg 540 tgggtgcgcc tgtacgtgct ggagctgtat tgcatcatcc tgggcctgcc cccttgtctg 600 aatatcctgc ggagaaagca gccccagctg accttcttta caatcgccct gcagtcttgt 660 cactatcaga ggctgccacc ccacatcctg tgggccacag gcctgaagtc tggaggatct 720 agcggaggat cctctggcag cgagacacca ggaacaagcg agtcagcaac accagagagc 780 agtggcggca gcagcggcgg cagcgccaag aacacaatta caaagacact gaagctgagg 840 atcgtgagac catacaacag cgctgaggtc gagaagattg tggctgatga aaagaacaac 900 agggaaaaga tcgccctcga gaagaacaag gataaggtga aggaggcctg ctctaagcac 960 ctgaaagtgg ccgcctactg caccacacag gtggagagga acgcctgtct gttttgtaaa 1020 gctcggaagc tggatgataa gttttaccag aagctgcggg gccagttccc cgatgccgtc 1080 ttttggcagg agattagcga gatcttcaga cagctgcaga agcaggccgc cgagatctac 1140 aaccagagcc tgatcgagct ctactacgag atcttcatca agggcaaggg cattgccaac 1200 gcctcctccg tggagcacta cctgagcgac gtgtgctaca caagagccgc cgagctcttt 1260 aagaacgccg ctatcgcttc cgggctgagg agcaagatta agagtaactt ccggctcaag 1320 gagctgaaga acatgaagag cggcctgccc actacaaaga gcgacaactt cccaattcca 1380 ctggtgaagc agaaggggggg ccagtacaca gggttcgaga tttccaacca caacagcgac 1440 tttattatta agatcccctt tggcaggtgg caggtcaaga aggagattga caagtacagg 1500 ccctgggaga agtttgattt cgagcaggtg cagaagagcc ccaagcctat ttccctgctg 1560 ctgtccacac agcggcggaa gaggaacaag gggtggtcta aggatgaggg gaccgaggcc 1620 gagattaaga aagtgatgaa cggcgactac cagacaagct acatcgaggt caagcggggc 1680 agtaagattg gcgagaagag cgcctggatg ctgaacctga gcattgacgt gccaaagatt 1740 gataagggcg tggatcccag catcatcgga gggatcgatg tgggggtcaa gagccccctc 1800 gtgtgcgcca tcaacaacgc cttcagcagg tacagcatct ccgataacga cctgttccac 1860 tttaacaaga agatgttcgc ccggcggagg attttgctca agaagaaccg gcacaagcgg 1920 gccggacacg gggccaagaa caagctcaag cccatcacta tcctgaccga gaagagcgag 1980 aggttcagga agaagctcat cgagagatgg gcctgcgaga tcgccgattt ctttattaag 2040 aacaaggtcg gaacagtgca gatggagaac ctcgagagca tgaagaggaa ggaggattcc 2100 tacttcaaca ttcggctgag ggggttctgg ccctacgctg agatgcagaa caagatgag 2160 tttaagctga agcagtacgg gattgagatc cggaaggtgg cccccaacaa caccagcaag 2220 acctgcagca agtgcgggca cctcaacaac tacttcaact tcgagtaccg gaagaagaac 2280 aagttcccac acttcaagtg cgagaagtgc aactttaagg agaacgccga ttacaacgcc 2340 gccctgaaca tcagcaaccc taagctgaag agcactaagg aggagcccag cggcgggagc 2400 ggcgggagcg gggggagcac taatctgagc gacatcattg agaaggagac tgggaaacag 2460 ctggtcattc aggagtccat cctgatgctg cctgaggagg tggaggaagt gatcggcaac 2520 aagccagagt ctgacatcct ggtgcacacc gcctacgacg agtccacaga tgagaatgtg 2580 atgctgctga cctctgacgc ccccgagtat aagccttggg ccctggtcat ccaggattct 2640 aacggcgaga ataagatcaa gatgctgagc ggaggatccg gaggatctgg aggcagcacc 2700 aacctgtctg acatcatcga gaaggagaca ggcaagcagc tggtcatcca ggagagcatc 2760 ctgatgctgc ccgaagaagt cgaagaagtg atcggaaaca agcctgagag cgatatcctg 2820 gtccataccg cctacgacga gagtaccgac gaaaatgtga tgctgctgac atccgacgcc 2880 ccagagtata agccctgggc tctggtcatc caggattcca acggagagaa caaaatcaaa 2940 atgctgtctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag 3000 aggaaagtc 3009 <210> 269 <211> 1586 <212> DNA <213> Artificial Sequence <220> <223> human codon-optimized Cas14a1 <400> 269 atggccaaga acacaattac aaagacactg aagctgagga tcgtgagacc atacaacagc 60 gctgaggtcg agaagattgt ggctgatgaa aagaacaaca gggaaaagat cgccctcgag 120 aagaacaagg ataaggtgaa ggaggcctgc tctaagcacc tgaaagtggc cgcctactgc 180 accacacagg tggagaggaa cgcctgtctg ttttgtaaag ctcggaagct ggatgataag 240 ttttaccaga agctgcgggg ccagttcccc gatgccgtct tttggcagga gattagcgag 300 atcttcagac agctgcagaa gcaggccgcc gagatctaca accagagcct gatcgagctc 360 tactacgaga tcttcatcaa gggcaagggc attgccaacg cctcctccgt ggagcactac 420 ctgagcgacg tgtgctacac aagagccgcc gagctcttta agaacgccgc tatcgcttcc 480 gggctgagga gcaagattaa gagtaacttc cggctcaagg agctgaagaa catgaagagc 540 ggcctgccca ctacaaagag cgacaacttc ccaattccac tggtgaagca gaaggggggc 600 cagtacacag ggttcgagat ttccaaccac aacagcgact ttattattaa gatccccttt 660 ggcaggtggc aggtcaagaa ggagattgac aagtacaggc cctgggagaa gtttgatttc 720 gagcaggtgc agaagagccc caagcctatt tccctgctgc tgtccacaca gcggcggaag 780 aggaacaagg ggtggtctaa ggatgagggg accgaggccg agattaagaa agtgatgaac 840 ggcgactacc agacaagcta catcgaggtc aagcggggca gtaagattgg cgagaagagc 900 gcctggatgc tgaacctgag cattgacgtg ccaaagattg ataagggcgt ggatcccagc 960 atcatcggag ggatcgatgt gggggtcaag agccccctcg tgtgcgccat caacaacgcc 1020 ttcagcaggt acagcatctc cgataacgac ctgttccact ttaacaagaa gatgttcgcc 1080 cggcggagga ttttgctcaa gaagaaccgg cacaagcggg ccggacacgg ggccaagaac 1140 aagctcaagc ccatcactat cctgaccgag aagagcgaga ggttcaggaa gaagctcatc 1200 gagagatggg cctgcgagat cgccgatttc tttattaaga acaaggtcgg aacagtgcag 1260 atggagaacc tcgagagcat gaagaggaag gaggattcct acttcaacat tcggctgagg 1320 gggttctggc cctacgctga gatgcagaac aagattgagt ttaagctgaa gcagtacggg 1380 attgagatcc ggaaggtggc ccccaacaac accagcaaga cctgcagcaa gtgcgggcac 1440 ctcaacaact acttcaactt cgagtaccgg aagaagaaca agttcccaca cttcaagtgc 1500 gagaagtgca actttaagga gaacgccgat tacaacgccg ccctgaacat cagcaaccct 1560 aagctgaaga gcactaagga ggagcc 1586 <210> 270 <211> 1607 <212> DNA <213> Artificial Sequence <220> <223> N-terminal NLS + Cas14a1 amino acid sequence <400> 270 atgccaaaga agaagcggaa agtcgccaag aacacaatta caaagacact gaagctgagg 60 atcgtgagac catacaacag cgctgaggtc gagaagattg tggctgatga aaagaacaac 120 agggaaaaga tcgccctcga gaagaacaag gataaggtga aggaggcctg ctctaagcac 180 ctgaaagtgg ccgcctactg caccacacag gtggagagga acgcctgtct gttttgtaaa 240 gctcggaagc tggatgataa gttttaccag aagctgcggg gccagttccc cgatgccgtc 300 ttttggcagg agattagcga gatcttcaga cagctgcaga agcaggccgc cgagatctac 360 aaccagagcc tgatcgagct ctactacgag atcttcatca agggcaaggg cattgccaac 420 gcctcctccg tggagcacta cctgagcgac gtgtgctaca caagagccgc cgagctcttt 480 aagaacgccg ctatcgcttc cgggctgagg agcaagatta agagtaactt ccggctcaag 540 gagctgaaga acatgaagag cggcctgccc actacaaaga gcgacaactt cccaattcca 600 ctggtgaagc agaaggggggg ccagtacaca gggttcgaga tttccaacca caacagcgac 660 tttattatta agatcccctt tggcaggtgg caggtcaaga aggagattga caagtacagg 720 ccctgggaga agtttgattt cgagcaggtg cagaagagcc ccaagcctat ttccctgctg 780 ctgtccacac agcggcggaa gaggaacaag gggtggtcta aggatgaggg gaccgaggcc 840 gagattaaga aagtgatgaa cggcgactac cagacaagct acatcgaggt caagcggggc 900 agtaagattg gcgagaagag cgcctggatg ctgaacctga gcattgacgt gccaaagatt 960 gataagggcg tggatcccag catcatcgga gggatcgatg tgggggtcaa gagccccctc 1020 gtgtgcgcca tcaacaacgc cttcagcagg tacagcatct ccgataacga cctgttccac 1080 tttaacaaga agatgttcgc ccggcggagg attttgctca agaagaaccg gcacaagcgg 1140 gccggacacg gggccaagaa caagctcaag cccatcacta tcctgaccga gaagagcgag 1200 aggttcagga agaagctcat cgagagatgg gcctgcgaga tcgccgattt ctttattaag 1260 aacaaggtcg gaacagtgca gatggagaac ctcgagagca tgaagaggaa ggaggattcc 1320 tacttcaaca ttcggctgag ggggttctgg ccctacgctg agatgcagaa caagatgag 1380 tttaagctga agcagtacgg gattgagatc cggaaggtgg cccccaacaa caccagcaag 1440 acctgcagca agtgcgggca cctcaacaac tacttcaact tcgagtaccg gaagaagaac 1500 aagttcccac acttcaagtg cgagaagtgc aactttaagg agaacgccga ttacaacgcc 1560 gccctgaaca tcagcaaccc taagctgaag agcactaagg aggagcc 1607 <210> 271 <211> 1607 <212> DNA <213> Artificial Sequence <220> <223> C-terminal NLS + Cas14a1 amino acid sequence <400> 271 atggccaaga acacaattac aaagacactg aagctgagga tcgtgagacc atacaacagc 60 gctgaggtcg agaagattgt ggctgatgaa aagaacaaca gggaaaagat cgccctcgag 120 aagaacaagg ataaggtgaa ggaggcctgc tctaagcacc tgaaagtggc cgcctactgc 180 accacacagg tggagaggaa cgcctgtctg ttttgtaaag ctcggaagct ggatgataag 240 ttttaccaga agctgcgggg ccagttcccc gatgccgtct tttggcagga gattagcgag 300 atcttcagac agctgcagaa gcaggccgcc gagatctaca accagagcct gatcgagctc 360 tactacgaga tcttcatcaa gggcaagggc attgccaacg cctcctccgt ggagcactac 420 ctgagcgacg tgtgctacac aagagccgcc gagctcttta agaacgccgc tatcgcttcc 480 gggctgagga gcaagattaa gagtaacttc cggctcaagg agctgaagaa catgaagagc 540 ggcctgccca ctacaaagag cgacaacttc ccaattccac tggtgaagca gaaggggggc 600 cagtacacag ggttcgagat ttccaaccac aacagcgact ttattattaa gatccccttt 660 ggcaggtggc aggtcaagaa ggagattgac aagtacaggc cctgggagaa gtttgatttc 720 gagcaggtgc agaagagccc caagcctatt tccctgctgc tgtccacaca gcggcggaag 780 aggaacaagg ggtggtctaa ggatgagggg accgaggccg agattaagaa agtgatgaac 840 ggcgactacc agacaagcta catcgaggtc aagcggggca gtaagattgg cgagaagagc 900 gcctggatgc tgaacctgag cattgacgtg ccaaagattg ataagggcgt ggatcccagc 960 atcatcggag ggatcgatgt gggggtcaag agccccctcg tgtgcgccat caacaacgcc 1020 ttcagcaggt acagcatctc cgataacgac ctgttccact ttaacaagaa gatgttcgcc 1080 cggcggagga ttttgctcaa gaagaaccgg cacaagcggg ccggacacgg ggccaagaac 1140 aagctcaagc ccatcactat cctgaccgag aagagcgaga ggttcaggaa gaagctcatc 1200 gagagatggg cctgcgagat cgccgatttc tttattaaga acaaggtcgg aacagtgcag 1260 atggagaacc tcgagagcat gaagaggaag gaggattcct acttcaacat tcggctgagg 1320 gggttctggc cctacgctga gatgcagaac aagattgagt ttaagctgaa gcagtacggg 1380 attgagatcc ggaaggtggc ccccaacaac accagcaaga cctgcagcaa gtgcgggcac 1440 ctcaacaact acttcaactt cgagtaccgg aagaagaaca agttcccaca cttcaagtgc 1500 gagaagtgca actttaagga gaacgccgat tacaacgccg ccctgaacat cagcaaccct 1560 aagctgaaga gcactaagga ggagccccaa agaagaagcg gaaagtc 1607 <210> 272 <211> 1628 <212> DNA <213> Artificial Sequence <220> <223> N/C-terminal NLS + Cas14a1 amino acid sequence <400> 272 atgccaaaga agaagcggaa agtcgccaag aacacaatta caaagacact gaagctgagg 60 atcgtgagac catacaacag cgctgaggtc gagaagattg tggctgatga aaagaacaac 120 agggaaaaga tcgccctcga gaagaacaag gataaggtga aggaggcctg ctctaagcac 180 ctgaaagtgg ccgcctactg caccacacag gtggagagga acgcctgtct gttttgtaaa 240 gctcggaagc tggatgataa gttttaccag aagctgcggg gccagttccc cgatgccgtc 300 ttttggcagg agattagcga gatcttcaga cagctgcaga agcaggccgc cgagatctac 360 aaccagagcc tgatcgagct ctactacgag atcttcatca agggcaaggg cattgccaac 420 gcctcctccg tggagcacta cctgagcgac gtgtgctaca caagagccgc cgagctcttt 480 aagaacgccg ctatcgcttc cgggctgagg agcaagatta agagtaactt ccggctcaag 540 gagctgaaga acatgaagag cggcctgccc actacaaaga gcgacaactt cccaattcca 600 ctggtgaagc agaaggggggg ccagtacaca gggttcgaga tttccaacca caacagcgac 660 tttattatta agatcccctt tggcaggtgg caggtcaaga aggagattga caagtacagg 720 ccctgggaga agtttgattt cgagcaggtg cagaagagcc ccaagcctat ttccctgctg 780 ctgtccacac agcggcggaa gaggaacaag gggtggtcta aggatgaggg gaccgaggcc 840 gagattaaga aagtgatgaa cggcgactac cagacaagct acatcgaggt caagcggggc 900 agtaagattg gcgagaagag cgcctggatg ctgaacctga gcattgacgt gccaaagatt 960 gataagggcg tggatcccag catcatcgga gggatcgatg tgggggtcaa gagccccctc 1020 gtgtgcgcca tcaacaacgc cttcagcagg tacagcatct ccgataacga cctgttccac 1080 tttaacaaga agatgttcgc ccggcggagg attttgctca agaagaaccg gcacaagcgg 1140 gccggacacg gggccaagaa caagctcaag cccatcacta tcctgaccga gaagagcgag 1200 aggttcagga agaagctcat cgagagatgg gcctgcgaga tcgccgattt ctttattaag 1260 aacaaggtcg gaacagtgca gatggagaac ctcgagagca tgaagaggaa ggaggattcc 1320 tacttcaaca ttcggctgag ggggttctgg ccctacgctg agatgcagaa caagatgag 1380 tttaagctga agcagtacgg gattgagatc cggaaggtgg cccccaacaa caccagcaag 1440 acctgcagca agtgcgggca cctcaacaac tacttcaact tcgagtaccg gaagaagaac 1500 aagttcccac acttcaagtg cgagaagtgc aactttaagg agaacgccga ttacaacgcc 1560 gccctgaaca tcagcaaccc taagctgaag agcactaagg aggagcccca aagaagaagc 1620 ggaaagtc 1628 <210> 273 <211> 3009 <212> DNA <213> Artificial Sequence <220> <223> Amino acid sequence (N terminal -Cytidine deaminase) <400> 273 atgccaaaga agaagcggaa agtctcctca gagactgggc ctgtcgccgt cgatccaacc 60 ctgcgccgcc ggattgaacc tcacgagttt gaagtgttct ttgacccccg ggagctgaga 120 aaggagacat gcctgctgta cgagatcaac tggggaggca ggcactccat ctggaggcac 180 acctctcaga acacaaataa gcacgtggag gtgaacttca tcgagaagtt taccacagag 240 cggtacttct gccccaatac cagatgtagc atcacatggt ttctgagctg gtccccttgc 300 ggagagtgta gcagggccat caccgagttc ctgtccagat atccacacgt gacactgttt 360 atctacatcg ccaggctgta tcaccacgca gacccaagga ataggcaggg cctgcgcgat 420 ctgatcagct ccggcgtgac catccagatc atgacagagc aggagtccgg ctactgctgg 480 cggaacttcg tgaattattc tcctagcaac gaggcccact ggcctaggta cccacacctg 540 tgggtgcgcc tgtacgtgct ggagctgtat tgcatcatcc tgggcctgcc cccttgtctg 600 aatatcctgc ggagaaagca gccccagctg accttcttta caatcgccct gcagtcttgt 660 cactatcaga ggctgccacc ccacatcctg tgggccacag gcctgaagtc tggaggatct 720 agcggaggat cctctggcag cgagacacca ggaacaagcg agtcagcaac accagagagc 780 agtggcggca gcagcggcgg cagcgccaag aacacaatta caaagacact gaagctgagg 840 atcgtgagac catacaacag cgctgaggtc gagaagattg tggctgatga aaagaacaac 900 agggaaaaga tcgccctcga gaagaacaag gataaggtga aggaggcctg ctctaagcac 960 ctgaaagtgg ccgcctactg caccacacag gtggagagga acgcctgtct gttttgtaaa 1020 gctcggaagc tggatgataa gttttaccag aagctgcggg gccagttccc cgatgccgtc 1080 ttttggcagg agattagcga gatcttcaga cagctgcaga agcaggccgc cgagatctac 1140 aaccagagcc tgatcgagct ctactacgag atcttcatca agggcaaggg cattgccaac 1200 gcctcctccg tggagcacta cctgagcgac gtgtgctaca caagagccgc cgagctcttt 1260 aagaacgccg ctatcgcttc cgggctgagg agcaagatta agagtaactt ccggctcaag 1320 gagctgaaga acatgaagag cggcctgccc actacaaaga gcgacaactt cccaattcca 1380 ctggtgaagc agaaggggggg ccagtacaca gggttcgaga tttccaacca caacagcgac 1440 tttattatta agatcccctt tggcaggtgg caggtcaaga aggagattga caagtacagg 1500 ccctgggaga agtttgattt cgagcaggtg cagaagagcc ccaagcctat ttccctgctg 1560 ctgtccacac agcggcggaa gaggaacaag gggtggtcta aggatgaggg gaccgaggcc 1620 gagattaaga aagtgatgaa cggcgactac cagacaagct acatcgaggt caagcggggc 1680 agtaagattg gcgagaagag cgcctggatg ctgaacctga gcattgacgt gccaaagatt 1740 gataagggcg tggatcccag catcatcgga gggatcgatg tgggggtcaa gagccccctc 1800 gtgtgcgcca tcaacaacgc cttcagcagg tacagcatct ccgataacga cctgttccac 1860 tttaacaaga agatgttcgc ccggcggagg attttgctca agaagaaccg gcacaagcgg 1920 gccggacacg gggccaagaa caagctcaag cccatcacta tcctgaccga gaagagcgag 1980 aggttcagga agaagctcat cgagagatgg gcctgcgaga tcgccgattt ctttattaag 2040 aacaaggtcg gaacagtgca gatggagaac ctcgagagca tgaagaggaa ggaggattcc 2100 tacttcaaca ttcggctgag ggggttctgg ccctacgctg agatgcagaa caagatgag 2160 tttaagctga agcagtacgg gattgagatc cggaaggtgg cccccaacaa caccagcaag 2220 acctgcagca agtgcgggca cctcaacaac tacttcaact tcgagtaccg gaagaagaac 2280 aagttcccac acttcaagtg cgagaagtgc aactttaagg agaacgccga ttacaacgcc 2340 gccctgaaca tcagcaaccc taagctgaag agcactaagg aggagcccag cggcgggagc 2400 ggcgggagcg gggggagcac taatctgagc gacatcattg agaaggagac tgggaaacag 2460 ctggtcattc aggagtccat cctgatgctg cctgaggagg tggaggaagt gatcggcaac 2520 aagccagagt ctgacatcct ggtgcacacc gcctacgacg agtccacaga tgagaatgtg 2580 atgctgctga cctctgacgc ccccgagtat aagccttggg ccctggtcat ccaggattct 2640 aacggcgaga ataagatcaa gatgctgagc ggaggatccg gaggatctgg aggcagcacc 2700 aacctgtctg acatcatcga gaaggagaca ggcaagcagc tggtcatcca ggagagcatc 2760 ctgatgctgc ccgaagaagt cgaagaagtg atcggaaaca agcctgagag cgatatcctg 2820 gtccataccg cctacgacga gagtaccgac gaaaatgtga tgctgctgac atccgacgcc 2880 ccagagtata agccctgggc tctggtcatc caggattcca acggagagaa caaaatcaaa 2940 atgctgtctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag 3000 aggaaagtc 3009 <210> 274 <211> 3009 <212> DNA <213> Artificial Sequence <220> <223> Amino acid sequence (C terminal -Cytidine deaminase) <400> 274 atgccaaaga agaagcggaa agtcgccaag aacacaatta caaagacact gaagctgagg 60 atcgtgagac catacaacag cgctgaggtc gagaagattg tggctgatga aaagaacaac 120 agggaaaaga tcgccctcga gaagaacaag gataaggtga aggaggcctg ctctaagcac 180 ctgaaagtgg ccgcctactg caccacacag gtggagagga acgcctgtct gttttgtaaa 240 gctcggaagc tggatgataa gttttaccag aagctgcggg gccagttccc cgatgccgtc 300 ttttggcagg agattagcga gatcttcaga cagctgcaga agcaggccgc cgagatctac 360 aaccagagcc tgatcgagct ctactacgag atcttcatca agggcaaggg cattgccaac 420 gcctcctccg tggagcacta cctgagcgac gtgtgctaca caagagccgc cgagctcttt 480 aagaacgccg ctatcgcttc cgggctgagg agcaagatta agagtaactt ccggctcaag 540 gagctgaaga acatgaagag cggcctgccc actacaaaga gcgacaactt cccaattcca 600 ctggtgaagc agaaggggggg ccagtacaca gggttcgaga tttccaacca caacagcgac 660 tttattatta agatcccctt tggcaggtgg caggtcaaga aggagattga caagtacagg 720 ccctgggaga agtttgattt cgagcaggtg cagaagagcc ccaagcctat ttccctgctg 780 ctgtccacac agcggcggaa gaggaacaag gggtggtcta aggatgaggg gaccgaggcc 840 gagattaaga aagtgatgaa cggcgactac cagacaagct acatcgaggt caagcggggc 900 agtaagattg gcgagaagag cgcctggatg ctgaacctga gcattgacgt gccaaagatt 960 gataagggcg tggatcccag catcatcgga gggatcgatg tgggggtcaa gagccccctc 1020 gtgtgcgcca tcaacaacgc cttcagcagg tacagcatct ccgataacga cctgttccac 1080 tttaacaaga agatgttcgc ccggcggagg attttgctca agaagaaccg gcacaagcgg 1140 gccggacacg gggccaagaa caagctcaag cccatcacta tcctgaccga gaagagcgag 1200 aggttcagga agaagctcat cgagagatgg gcctgcgaga tcgccgattt ctttattaag 1260 aacaaggtcg gaacagtgca gatggagaac ctcgagagca tgaagaggaa ggaggattcc 1320 tacttcaaca ttcggctgag ggggttctgg ccctacgctg agatgcagaa caagatgag 1380 tttaagctga agcagtacgg gattgagatc cggaaggtgg cccccaacaa caccagcaag 1440 acctgcagca agtgcgggca cctcaacaac tacttcaact tcgagtaccg gaagaagaac 1500 aagttcccac acttcaagtg cgagaagtgc aactttaagg agaacgccga ttacaacgcc 1560 gccctgaaca tcagcaaccc taagctgaag agcactaagg aggagccctc tggaggatct 1620 agcggaggat cctctggcag cgagacacca ggaacaagcg agtcagcaac accagagagc 1680 agtggcggca gcagcggcgg cagctcctca gagactgggc ctgtcgccgt cgatccaacc 1740 ctgcgccgcc ggattgaacc tcacgagttt gaagtgttct ttgacccccg ggagctgaga 1800 aaggagacat gcctgctgta cgagatcaac tggggaggca ggcactccat ctggaggcac 1860 acctctcaga acacaaataa gcacgtggag gtgaacttca tcgagaagtt taccacagag 1920 cggtacttct gccccaatac cagatgtagc atcacatggt ttctgagctg gtccccttgc 1980 ggagagtgta gcagggccat caccgagttc ctgtccagat atccacacgt gacactgttt 2040 atctacatcg ccaggctgta tcaccacgca gacccaagga ataggcaggg cctgcgcgat 2100 ctgatcagct ccggcgtgac catccagatc atgacagagc aggagtccgg ctactgctgg 2160 cggaacttcg tgaattattc tcctagcaac gaggcccact ggcctaggta cccacacctg 2220 tgggtgcgcc tgtacgtgct ggagctgtat tgcatcatcc tgggcctgcc cccttgtctg 2280 aatatcctgc ggagaaagca gccccagctg accttcttta caatcgccct gcagtcttgt 2340 cactatcaga ggctgccacc ccacatcctg tgggccacag gcctgaagag cggcgggagc 2400 ggcgggagcg gggggagcac taatctgagc gacatcattg agaaggagac tgggaaacag 2460 ctggtcattc aggagtccat cctgatgctg cctgaggagg tggaggaagt gatcggcaac 2520 aagccagagt ctgacatcct ggtgcacacc gcctacgacg agtccacaga tgagaatgtg 2580 atgctgctga cctctgacgc ccccgagtat aagccttggg ccctggtcat ccaggattct 2640 aacggcgaga ataagatcaa gatgctgagc ggaggatccg gaggatctgg aggcagcacc 2700 aacctgtctg acatcatcga gaaggagaca ggcaagcagc tggtcatcca ggagagcatc 2760 ctgatgctgc ccgaagaagt cgaagaagtg atcggaaaca agcctgagag cgatatcctg 2820 gtccataccg cctacgacga gagtaccgac gaaaatgtga tgctgctgac atccgacgcc 2880 ccagagtata agccctgggc tctggtcatc caggattcca acggagagaa caaaatcaaa 2940 atgctgtctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag 3000 aggaaagtc 3009 <210> 275 <211> 2823 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence (N terminal -Adenine deaminase) <400> 275 atgtccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 60 agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 120 atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 180 atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 240 tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta tgattcactc ccgcattgga 300 cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 360 ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 420 tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 480 aaagcacaat cctctactga ctctggtggt tcttctggtg gttctagcgg cagcgagact 540 cccgggacct cagagtccgc cacacccgaa agttctggtg gttcttctgg tggttcttcc 600 gaagtcgagt tttcccatga gtactggatg agacacgcat tgactctcgc aaagagggct 660 cgagatgaac gcgaggtgcc cgtgggggca gtactcgtgc tcaacaatcg cgtaatcggc 720 gaaggttgga atagggcaat cggactccac gaccccactg cacatgcgga aatcatggcc 780 cttcgacagg gagggcttgt gatgcagaat tatcgactta tcgatgcgac gctgtacgtc 840 acgtttgaac cttgcgtaat gtgcgcggga gctatgattc actcccgcat tggacgagtt 900 gtattcggtg ttcgcaacgc caagacgggt gccgcaggtt cactgatgga cgtgctgcat 960 tacccaggca tgaaccaccg ggtagaaatc acagaaggca tattggcgga cgaatgtgcg 1020 gcgctgttgt gttacttttt tcgcatgccc aggcaggtct ttaacgccca gaaaaaagca 1080 caatcctcta ctgactctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1140 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgccaagaac 1200 acaattacaa agacactgaa gctgaggatc gtgagaccat acaacagcgc tgaggtcgag 1260 aagattgtgg ctgatgaaaa gaacaacagg gaaaagatcg ccctcgagaa gaacaaggat 1320 aaggtgaagg aggcctgctc taagcacctg aaagtggccg cctactgcac cacacaggtg 1380 gagaggaacg cctgtctgtt ttgtaaagct cggaagctgg atgataagtt ttaccagaag 1440 ctgcggggcc agttccccga tgccgtcttt tggcaggaga ttagcgagat cttcagacag 1500 ctgcagaagc aggccgccga gatctacaac cagagcctga tcgagctcta ctacgagatc 1560 ttcatcaagg gcaagggcat tgccaacgcc tcctccgtgg agcactacct gagcgacgtg 1620 tgctacacaa gagccgccga gctctttaag aacgccgcta tcgcttccgg gctgaggagc 1680 aagattaaga gtaacttccg gctcaaggag ctgaagaaca tgaagagcgg cctgcccact 1740 acaaagagcg acaacttccc aattccactg gtgaagcaga aggggggcca gtacacaggg 1800 ttcgagattt ccaaccacaa cagcgacttt attattaaga tcccctttgg caggtggcag 1860 gtcaagaagg agattgacaa gtacaggccc tgggagaagt ttgatttcga gcaggtgcag 1920 aagagcccca agcctatttc cctgctgctg tccacacagc ggcggaagag gaacaagggg 1980 tggtctaagg atgaggggac cgaggccgag attaagaaag tgatgaacgg cgactaccag 2040 acaagctaca tcgaggtcaa gcggggcagt aagattggcg agaagagcgc ctggatgctg 2100 aacctgagca ttgacgtgcc aaagattgat aagggcgtgg atcccagcat catcggaggg 2160 atcgatgtgg gggtcaagag ccccctcgtg tgcgccatca acaacgcctt cagcaggtac 2220 agcatctccg ataacgacct gttccacttt aacaagaaga tgttcgcccg gcggaggatt 2280 ttgctcaaga agaaccggca caagcgggcc ggacacgggg ccaagaacaa gctcaagccc 2340 atcactatcc tgaccgagaa gagcgagagg ttcaggaaga agctcatcga gagatgggcc 2400 tgcgagatcg ccgatttctt tattaagaac aaggtcggaa cagtgcagat ggagaacctc 2460 gagagcatga agaggaagga ggattcctac ttcaacattc ggctgagggg gttctggccc 2520 tacgctgaga tgcagaacaa gattgagttt aagctgaagc agtacgggat tgagatccgg 2580 aaggtggccc ccaacaacac cagcaagacc tgcagcaagt gcgggcacct caacaactac 2640 ttcaacttcg agtaccggaa gaagaacaag ttcccacact tcaagtgcga gaagtgcaac 2700 tttaaggaga acgccgatta caacgccgcc ctgaacatca gcaaccctaa gctgaagagc 2760 actaaggagg agcccaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 2820 aag 2823 <210> 276 <211> 2823 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence (C terminal -Adenine deaminase) <400> 276 atggccaaga acacaattac aaagacactg aagctgagga tcgtgagacc atacaacagc 60 gctgaggtcg agaagattgt ggctgatgaa aagaacaaca gggaaaagat cgccctcgag 120 aagaacaagg ataaggtgaa ggaggcctgc tctaagcacc tgaaagtggc cgcctactgc 180 accacacagg tggagaggaa cgcctgtctg ttttgtaaag ctcggaagct ggatgataag 240 ttttaccaga agctgcgggg ccagttcccc gatgccgtct tttggcagga gattagcgag 300 atcttcagac agctgcagaa gcaggccgcc gagatctaca accagagcct gatcgagctc 360 tactacgaga tcttcatcaa gggcaagggc attgccaacg cctcctccgt ggagcactac 420 ctgagcgacg tgtgctacac aagagccgcc gagctcttta agaacgccgc tatcgcttcc 480 gggctgagga gcaagattaa gagtaacttc cggctcaagg agctgaagaa catgaagagc 540 ggcctgccca ctacaaagag cgacaacttc ccaattccac tggtgaagca gaaggggggc 600 cagtacacag ggttcgagat ttccaaccac aacagcgact ttattattaa gatccccttt 660 ggcaggtggc aggtcaagaa ggagattgac aagtacaggc cctgggagaa gtttgatttc 720 gagcaggtgc agaagagccc caagcctatt tccctgctgc tgtccacaca gcggcggaag 780 aggaacaagg ggtggtctaa ggatgagggg accgaggccg agattaagaa agtgatgaac 840 ggcgactacc agacaagcta catcgaggtc aagcggggca gtaagattgg cgagaagagc 900 gcctggatgc tgaacctgag cattgacgtg ccaaagattg ataagggcgt ggatcccagc 960 atcatcggag ggatcgatgt gggggtcaag agccccctcg tgtgcgccat caacaacgcc 1020 ttcagcaggt acagcatctc cgataacgac ctgttccact ttaacaagaa gatgttcgcc 1080 cggcggagga ttttgctcaa gaagaaccgg cacaagcggg ccggacacgg ggccaagaac 1140 aagctcaagc ccatcactat cctgaccgag aagagcgaga ggttcaggaa gaagctcatc 1200 gagagatggg cctgcgagat cgccgatttc tttattaaga acaaggtcgg aacagtgcag 1260 atggagaacc tcgagagcat gaagaggaag gaggattcct acttcaacat tcggctgagg 1320 gggttctggc cctacgctga gatgcagaac aagattgagt ttaagctgaa gcagtacggg 1380 attgagatcc ggaaggtggc ccccaacaac accagcaaga cctgcagcaa gtgcgggcac 1440 ctcaacaact acttcaactt cgagtaccgg aagaagaaca agttcccaca cttcaagtgc 1500 gagaagtgca actttaagga gaacgccgat tacaacgccg ccctgaacat cagcaaccct 1560 aagctgaaga gcactaagga ggagccctct ggaggatcta gcggaggatc ctctggcagc 1620 gagacaccag gaacaagcga gtcagcaaca ccagagagca gtggcggcag cagcggcggc 1680 agctccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 1740 agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 1800 atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 1860 atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 1920 tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta tgattcactc ccgcattgga 1980 cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 2040 ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 2100 tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 2160 aaagcacaat cctctactga ctctggtggt tcttctggtg gttctagcgg cagcgagact 2220 cccgggacct cagagtccgc cacacccgaa agttcaggtg gatcttcagg tggatcttcg 2280 gaagtggaat tttcgcacga gtattggatg aggcacgctt taactctcgc taagagagca 2340 cgagacgaac gggaagtgcc ggttggggct gtcctcgtac tcaataatcg agttatcgga 2400 gaaggctgga acagggcaat cggactccac gatcccacag ctcatgccga gataatggcg 2460 cttcgacaag gaggcctagt catgcaaaat tatcgtctta ttgacgcgac cctctacgtg 2520 acctttgagc catgcgttat gtgtgcgggt gcaatgatac attcccggat aggacgtgta 2580 gtatttggag ttcgcaacgc gaagaccggt gcggctggtt ctctcatgga tgtcctgcac 2640 taccctggga tgaatcaccg cgttgaaatc actgaaggca ttttggccga tgaatgcgcg 2700 gccctgttat gttacttttt tcgcatgccc aggcaggtct ttaacgcaca gaagaaagcc 2760 caatcgtcca ctgataaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 2820 aag 2823 <210> 277 <211> 7 <212> PRT <213> simian virus 40 <400> 277 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 278 <211> 16 <212> PRT <213> mus musculus <400> 278 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 279 <211> 9 <212> PRT <213> homo sapiens <400> 279 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 280 <211> 11 <212> PRT <213> homo sapiens <400> 280 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10 <210> 281 <211> 38 <212> PRT <213> mus musculus <400> 281 Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35 <210> 282 <211> 42 <212> PRT <213> Homo sapiens <400> 282 Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu Lys Arg Arg Asn Val 35 40 <210> 283 <211> 8 <212> PRT <213> Homo sapiens <400> 283 Val Ser Arg Lys Arg Pro Arg Pro 1 5 <210> 284 <211> 8 <212> PRT <213> homo sapiens <400> 284 Pro Pro Lys Lys Ala Arg Glu Asp 1 5 <210> 285 <211> 8 <212> PRT <213> homo sapiens <400> 285 Pro Gln Pro Lys Lys Lys Pro Leu 1 5 <210> 286 <211> 12 <212> PRT <213> mus musculus <400> 286 Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 <210> 287 <211> 5 <212> PRT <213> Influenza virus <400> 287 Asp Arg Leu Arg Arg 1 5 <210> 288 <211> 7 <212> PRT <213> influenza virus <400> 288 Pro Lys Gln Lys Lys Arg Lys 1 5 <210> 289 <211> 10 <212> PRT <213> Hepatitis D virus <400> 289 Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 <210> 290 <211> 10 <212> PRT <213> mus musculus <400> 290 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> 291 <211> 20 <212> PRT <213> homo sapiens <400> 291 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 <210> 292 <211> 17 <212> PRT <213> Homo sapiens <400> 292 Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys <210> 293 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 293 aacaaauuca uuuugaaacg aaugaagga 29 <210> 294 <211> 31 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 294 aacaaauuca uuuuugaaaa cgaaugaagg a 31 <210> 295 <211> 33 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 295 aacaaauuca uuuuucgaaa gacgaaugaa gga 33 <210> 296 <211> 35 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 296 aacaaauuca uuuuuccgaa aagacgaaug aagga 35 <210> 297 <211> 37 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 297 aacaaauuca uuuuuccuga aauagacgaa ugaagga 37 <210> 298 <211> 39 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 298 aacaaauuca uuuuuccucg aaaauagacg aaugaagga 39 <210> 299 <211> 41 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 299 aacaaauuca uuuuuccucu gaaaaauaga cgaaugaagg a 41 <210> 300 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 300 aacaaauuca uuuuuccucu cgaaagaaua gacgaaugaa gga 43 <210> 301 <211> 45 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 301 aacaaauuca uuuuuccucu ccgaaacgaa uagacgaaug aagga 45 <210> 302 <211> 47 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 302 aacaaauuca uuuuuccucu ccagaaaccg aauagacgaa ugaagga 47 <210> 303 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 303 aacaaauuca uuuuuccucu ccaagaaacc cgaauagacg aaugaagga 49 <210> 304 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 304 aacaaauuca uuuuuccucu ccaaugaaaa cccgaauaga cgaaugaagg a 51 <210> 305 <211> 53 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 305 aacaaauuca uuuuuccucu ccaauugaaa aacccgaaua gacgaaugaa gga 53 <210> 306 <211> 55 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 306 aacaaauuca uuuuuccucu ccaauucgaa agaacccgaa uagacgaaug aagga 55 <210> 307 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 307 aacaaauuca uuuuuccucu ccaauucuga aaagaacccg aauagacgaa ugaagga 57 <210> 308 <211> 59 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 308 aacaaauuca uuuuuccucu ccaauucugg aaacagaacc cgaauagacg aaugaagga 59 <210> 309 <211> 61 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 309 aacaaauuca uuuuuccucu ccaauucugc gaaagcagaa cccgaauaga cgaaugaagg 60 a 61 <210> 310 <211> 63 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 310 aacaaauuca uuuuuccucu ccaauucugc agaaaugcag aacccgaaua gacgaaugaa 60 gga 63 <210> 311 <211> 65 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 311 aacaaauuca uuuuuccucu ccaauucugc acgaaauugc agaacccgaa uagacgaaug 60 aagga 65 <210> 312 <211> 67 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 312 aacaaauuca uuuuuccucu ccaauucugc acagaaaguu gcagaacccg aauagacgaa 60 ugaagga 67 <210> 313 <211> 66 <212> RNA <213> Artificial Sequence <220> <223> engineered (4th region + Linker + 5th region) of WT gRNA <400> 313 aacaaauuca uuuuuccucu ccaauucugc acagaaauug cagaacccga auagacgaau 60 gaagga 66 <210> 314 <211> 68 <212> RNA <213> Artificial Sequence <220> <223> (4th region + Linker + 5th region) of WT gRNA <400> 314 aacaaauuca uuuuuccucu ccaauucugc acaagaaagu ugcagaaccc gaauagacga 60 augaagga 68 <210> 315 <211> 202 <212> RNA <213> Artificial Sequence <220> <223> wild-type of Cas12f1 tracrRNA + GAAA + wild-type of Cas12f1 crRNA repeat <400> 315 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu uuccucucca auucugcaca agaaaguugc agaacccgaa 180 uagacgaaug aaggaaugca ac 202 <210> 316 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 316 caagaaaguu 10 <210> 317 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 317 acaagaaagu ug 12 <210> 318 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 318 cacaagaaag uugc 14 <210> 319 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 319 gcacaagaaa guugca 16 <210> 320 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 320 ugcacaagaa aguugcag 18 <210> 321 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 321 cugcacaaga aaguugcaga 20 <210> 322 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 322 ucugcacaag aaaguugcag aa 22 <210> 323 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 323 uucugcacaa gaaaguugca gaac 24 <210> 324 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 324 auucugcaca agaaaguugc agaacc 26 <210> 325 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 325 aauucugcac aagaaaguug cagaaccc 28 <210> 326 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 326 caauucugca caagaaaguu gcagaacccg 30 <210> 327 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 327 ccaauucugc acaagaaagu ugcagaaccc ga 32 <210> 328 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 328 uccaauucug cacaagaaag uugcagaacc cgaa 34 <210> 329 <211> 36 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 329 cuccaauucu gcacaagaaa guugcagaac ccgaau 36 <210> 330 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 330 ucuccaauuc ugcacaagaa aguugcagaa cccgaaua 38 <210> 331 <211> 40 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 331 cucuccaauu cugcacaaga aaguugcaga acccgaauag 40 <210> 332 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 332 ccucuccaau ucugcacaag aaaguugcag aacccgaaua ga 42 <210> 333 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 333 ucucuccaa uucugcacaa gaaaguugca gaacccgaau agac 44 <210> 334 <211> 46 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 334 uuccucucca auucugcaca agaaaguugc agaacccgaa uagacg 46 <210> 335 <211> 48 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 335 uuuccucucc aauucugcac aagaaaguug cagaacccga auagacga 48 <210> 336 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 336 uuuuccucuc caauucugca caagaaaguu gcagaacccg aauagacgaa 50 <210> 337 <211> 52 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 337 uuuuuccucu ccaauucugc acaagaaagu ugcagaaccc gaauagacga au 52 <210> 338 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 338 auuuuuccuc uccaauucug cacaagaaag uugcagaacc cgaauagacg aaug 54 <210> 339 <211> 56 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 339 cauuuuuccu cuccaauucu gcacaagaaa guugcagaac ccgaauagac gaauga 56 <210> 340 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 340 ucauuuuucc ucuccaauuc ugcacaagaa aguugcagaa cccgaauaga cgaaugaa 58 <210> 341 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 341 57 ucauuuuucc ucuccaauuc ugcacaagaa aguugcagaa cccgaauaga cgaauga 57 <210> 342 <211> 59 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, WT <400> 342 uucauuuuuc cucuccaauu cugcacaaga aaguugcaga acccgaauag acgaaugaa 59 <210> 343 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 343 uucauuuuuc 10 <210> 344 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 344 uucauuuuuc c 11 <210> 345 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 345 uucauuuuuc cu 12 <210> 346 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 346 cuc 13 <210> 347 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 347 cucu 14 <210> 348 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 348 uucauuuuuc cucuc 15 <210> 349 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 349 uucauuuuuc cucucc 16 <210> 350 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 350 uucauuuuuc cucucca 17 <210> 351 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 351 uucauuuuuc cucuccaa 18 <210> 352 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 352 uucauuuuuc cucuccaau 19 <210> 353 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 353 uucauuuuuc cucuccaauu 20 <210> 354 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 354 uucauuuuuc cucuccaauu c 21 <210> 355 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 355 uucauuuuuc cucuccaauu cu 22 <210> 356 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 356 uucauuuuuc cucuccaauu cug 23 <210> 357 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 357 uucauuuuuc cucuccaauu cugc 24 <210> 358 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 358 uucauuuuuc cucuccaauu cugca 25 <210> 359 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 359 uucauuuuuc cucuccaauu cugcac 26 <210> 360 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 360 uucauuuuuc cucuccaauu cugcaca 27 <210> 361 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 4th region, WT <400> 361 uucauuuuuc cucuccaauu cugcacaa 28 <210> 362 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 362 gacgaaugaa 10 <210> 363 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 363 agacgaauga a 11 <210> 364 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 364 uagacgaaug aa 12 <210> 365 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 365 auagacgaau gaa 13 <210> 366 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 366 aauagacgaa ugaa 14 <210> 367 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 367 gaauagacga augaa 15 <210> 368 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 368 cgaauagacg aaugaa 16 <210> 369 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 369 ccgaauagac gaaugaa 17 <210> 370 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 370 cccgaauaga cgaaugaa 18 <210> 371 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 371 acccgaauag acgaaugaa 19 <210> 372 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 372 aacccgaaua gacgaaugaa 20 <210> 373 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 373 gaacccgaau agacgaauga a 21 <210> 374 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 374 agaacccgaa uagacgaaug aa 22 <210> 375 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 375 cagaacccga auagacgaau gaa 23 <210> 376 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 376 gcagaacccg aauagacgaa ugaa 24 <210> 377 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 377 ugcagaaccc gaauagacga augaa 25 <210> 378 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 378 uugcagaacc cgaauagacg aaugaa 26 <210> 379 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> deleted part of 5th region, WT <400> 379 guugcagaac ccgaauagac gaaugaa 27 <210> 380 <211> 181 <212> RNA <213> Artificial Sequence <220> <223> Comparative Example 1.1 <400> 380 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu gaaagaauga aggaaugcaa ccacacacac agugggcuac 180 c 181 <210> 381 <211> 174 <212> RNA <213> Artificial Sequence <220> <223> Example 1.1 <400> 381 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuugaaagaa ugaaggaaug caaccacaca cacagugggc uacc 174 <210> 382 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 1.2 <400> 382 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuugaaa gaaugaagga augcaaccac acacacagug ggcuacc 167 <210> 383 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> Example 1.3 <400> 383 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 gaaagaauga aggaaugcaa ccacacacac agugggcuac c 161 <210> 384 <211> 175 <212> RNA <213> Artificial Sequence <220> <223> Example 1.4 <400> 384 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaaccacac acacaguggg cuacc 175 <210> 385 <211> 170 <212> RNA <213> Artificial Sequence <220> <223> Example 1.5 <400> 385 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac cacacacaca gugggcuacc 170 <210> 386 <211> 158 <212> RNA <213> Artificial Sequence <220> <223> Example 1.6 <400> 386 cuucacugau aaaguggaga accgcuucac cauuaugag ugaagguggg cugcuugcau 60 cagccuaaug ucgagaagug cuuucuucgg aaaguaaccc ucgaaacaaa uucauuugaa 120 agaaugaagg aaugcaacca cacacacagu gggcuacc 158 <210> 387 <211> 177 <212> RNA <213> Artificial Sequence <220> <223> Example 1.7 <400> 387 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauga aaaugaagga augcaaccac acacacagug ggcuacc 177 <210> 388 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> Example 1.8 <400> 388 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgaaa gaaggaaugc aaccacacac acagugggcu acc 173 <210> 389 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 1.9 <400> 389 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaagaaagga augcaaccac acacacagug ggcuacc 167 <210> 390 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> Example 1.10 <400> 390 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac 120 cacacacaca gugggcuacc 140 <210> 391 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> Example 1.11 <400> 391 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaaccac acacacagug ggcuacc 147 <210> 392 <211> 146 <212> RNA <213> Artificial Sequence <220> <223> Example 1.12 <400> 392 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa 120 ugcaaccaca cacacagugg gcuacc 146 <210> 393 <211> 126 <212> RNA <213> Artificial Sequence <220> <223> Example 1.13 <400> 393 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa ugcaaccaca cacacagugg 120 gcuacc 126 <210> 394 <211> 181 <212> RNA <213> Artificial Sequence <220> <223> Comparative Example 2.1 <400> 394 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu gaaagaauga aggaaugcaa ccauccccag gacacacaca 180 c 181 <210> 395 <211> 174 <212> RNA <213> Artificial Sequence <220> <223> Example 2.1 <400> 395 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuugaaagaa ugaaggaaug caaccauccc caggacacac acac 174 <210> 396 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 2.2 <400> 396 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuugaaa gaaugaagga augcaaccau ccccaggaca cacacac 167 <210> 397 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> Example 2.3 <400> 397 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 gaaagaauga aggaaugcaa ccauccccag gacacacaca c 161 <210> 398 <211> 175 <212> RNA <213> Artificial Sequence <220> <223> Example 2.4 <400> 398 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaaccaucc ccaggacaca cacac 175 <210> 399 <211> 170 <212> RNA <213> Artificial Sequence <220> <223> Example 2.5 <400> 399 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac cauccccagg acacacacac 170 <210> 400 <211> 160 <212> RNA <213> Artificial Sequence <220> <223> Example 2.6 <400> 400 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug 120 aaagaaugaa ggaaugcaac cauccccagg acacacacac 160 <210> 401 <211> 177 <212> RNA <213> Artificial Sequence <220> <223> Example 2.7 <400> 401 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauga aaaugaagga augcaaccau ccccaggaca cacacac 177 <210> 402 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> Example 2.8 <400> 402 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgaaa gaaggaaugc aaccaucccc aggacacaca cac 173 <210> 403 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 2.9 <400> 403 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaagaaagga augcaaccau ccccaggaca cacacac 167 <210> 404 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> Example 2.10 <400> 404 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac 120 cauccccagg acacacacac 140 <210> 405 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> Example 2.11 <400> 405 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaaccau ccccaggaca cacacac 147 <210> 406 <211> 146 <212> RNA <213> Artificial Sequence <220> <223> Example 2.12 <400> 406 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa 120 ugcaaccauc cccaggacac acacac 146 <210> 407 <211> 126 <212> RNA <213> Artificial Sequence <220> <223> Example 2.13 <400> 407 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa ugcaaccauc cccaggacac 120 acacac 126 <210> 408 <211> 181 <212> RNA <213> Artificial Sequence <220> <223> Comparative Example 3.1 <400> 408 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu gaaagaauga aggaaugcaa cagaacacau accccugggc 180 c 181 <210> 409 <211> 174 <212> RNA <213> Artificial Sequence <220> <223> Example 3.1 <400> 409 gauaaagugg agaaccgcuu caccaaaagc ugucccuuag gggauuagaa cuugagugaa 60 ggugggcugc uugcaucagc cuaaugucga gaagugcuuu cuucggaaag uaacccucga 120 aacaaauuca uuugaaagaa ugaaggaaug caacagaaca cauaccccug ggcc 174 <210> 410 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 3.2 <400> 410 uggagaaccg cuucaccaaa agcugucccu uaggggauua gaacuugagu gaaggugggc 60 ugcuugcauc agccuaaugu cgagaagugc uuucuucgga aaguaacccu cgaaacaaau 120 ucauuugaaa gaaugaagga augcaacaga acacauaccc cugggcc 167 <210> 411 <211> 161 <212> RNA <213> Artificial Sequence <220> <223> Example 3.3 <400> 411 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucauuu 120 gaaagaauga aggaaugcaa cagaacacau accccugggc c 161 <210> 412 <211> 175 <212> RNA <213> Artificial Sequence <220> <223> Example 3.4 <400> 412 cuucacugau aaaguggaga accgcuucac caaaagcugu uuagauuaga acuugaguga 60 aggugggcug cuugcaucag ccuaaugucg agaagugcuu ucuucggaaa guaacccucg 120 aaacaaauuc auuugaaaga augaaggaau gcaacagaac acauaccccu gggcc 175 <210> 413 <211> 170 <212> RNA <213> Artificial Sequence <220> <223> Example 3.5 <400> 413 cuucacugau aaaguggaga accgcuucac caaaagcuuu agagaacuug agugaaggug 60 ggcugcuugc aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca 120 aauucauuug aaagaaugaa ggaaugcaac agaacacaua ccccugggcc 170 <210> 414 <211> 160 <212> RNA <213> Artificial Sequence <220> <223> Example 3.6 <400> 414 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug 120 aaagaaugaa ggaaugcaac agaacacaua ccccugggcc 160 <210> 415 <211> 177 <212> RNA <213> Artificial Sequence <220> <223> Example 3.7 <400> 415 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauga aaaugaagga augcaacaga acacauaccc cugggcc 177 <210> 416 <211> 173 <212> RNA <213> Artificial Sequence <220> <223> Example 3.8 <400> 416 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgaaa gaaggaaugc aacagaacac auaccccugg gcc 173 <210> 417 <211> 167 <212> RNA <213> Artificial Sequence <220> <223> Example 3.9 <400> 417 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaagaaagga augcaacaga acacauaccc cugggcc 167 <210> 418 <211> 140 <212> RNA <213> Artificial Sequence <220> <223> Example 3.10 <400> 418 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac 120 agaacacaua ccccugggcc 140 <210> 419 <211> 147 <212> RNA <213> Artificial Sequence <220> <223> Example 3.11 <400> 419 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaacaga acacauaccc cugggcc 147 <210> 420 <211> 146 <212> RNA <213> Artificial Sequence <220> <223> Example 3.12 <400> 420 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa 120 ugcaacagaa cacauacccc ugggcc 146 <210> 421 <211> 126 <212> RNA <213> Artificial Sequence <220> <223> Example 3.13 <400> 421 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aagaaaggaa ugcaacagaa cacauacccc 120 ugggcc 126 <210> 422 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> protospacer sequence for targeting DY2 <400> 422 cacacacaca gtgggctacc 20 <210> 423 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> protospacer sequence for targeting DY10 <400> 423 catccccagg acacacacac 20 <210> 424 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> protospacer sequence for targeting Intergenic-22 <400> 424 agaacacata cccctgggcc 20 <210> 425 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, MF <400> 425 uucgaaagaa 10 <210> 426 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, MF <400> 426 uucagaaaug aa 12 <210> 427 <211> 14 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, MF <400> 427 uucaugaaaa ugaa 14 <210> 428 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, MF <400> 428 uucauugaaa aaugaa 16 <210> 429 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> Linker variation, MF <400> 429 uucauuugaa agaaugaa 18 <210> 430 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> Second region, 13bp deletion <400> 430 ccgcuucacu uagagugaag gugg 24 <210> 431 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> Second region, 12bp deletion <400> 431 ccgcuucacc uuaggaguga aggugg 26 <210> 432 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> second region, 5' remains <400> 432 ccgcuucac 9 <210> 433 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> second region, 3' remains <400> 433 agugaaggug g 11 <210> 434 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region <400> 434 aaaagcuguc ccuuagggga uuagaacuu 29 <210> 435 <211> 31 <212> RNA <213> Artificial Sequence <220> <223> intermediate sequence of the second region <400> 435 caaaagcugu cccuuagggg auuagaacuu g 31 <210> 436 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> Example 1.14 <400> 436 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaagaaa ggaaugcaac cacacacaca gugggcuacc 120 120 <210> 437 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> Example 2.14 <400> 437 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaagaaa ggaaugcaac cauccccagg acacacacac 120 120 <210> 438 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> Example 3.14 <400> 438 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaagaaa ggaaugcaac agaacacaua ccccugggcc 120 120 <210> 439 <211> 120 <212> RNA <213> Artificial Sequence <220> <223> engineered sgRNA <400> 439 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaagaaa ggaaugcaac nnnnnnnnnn nnnnnnnnnn 120 120 <210> 440 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Upper deleted part of second region <400> 440 aaaagcuguc cc 12 <210> 441 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Lower deleted part of second region <400> 441 caaaagcugu ccc 13

Claims (33)

Engineered guide RNAs for the CRISPR/Cas12f1 system, including:
engineered scaffold area; and
spacer;
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and
5'-AUGCAAC-3'. Engineered guide RNAs for the CRISPR/Cas12f1 system, including:
engineered scaffold area; and
spacer; and
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUUCCUCUCCCAAAAUUCGAGAACCAAUGAAUGAAUUCGAUGAAGAAUGAAUGAAUGAUGAAGUGAGAAUGAAUGAAUGAUGAUGAGAAUGAAUGAAUGAUGAAUGAAGAAU characterized by that the sequence differs by the sequence
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) No. 117), 5'-AACAAAUUCAUUUUGAAACGAAUGAAGGA-3' (SEQ ID NO: 293), 5'-AACAAAUUCAUUUUUGAAAACGAAUGAAGGA-3' (SEQ ID NO: 294), 5'-AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAAU-GAAAAAGGAUGA (SEQ ID NO: 296), 5'-AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 297), 5'-AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 298), 5'-AACAAAUUCAUUCAUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAACUGAAUGAAUUUUUCCUCUGAAUGAAGAAUUCA 3' (SEQ ID NO: 300), 5'-AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 301), 5'-AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAGU-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAU AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 304), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 305), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 306), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 307), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 308 ), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 309), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 310), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 311), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열 312), 5'-AACAAAUUCAUUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 313), and 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO:314) selected from the group consisting of; and
5'-AUGCAAC-3'. According to claim 1, wherein the sequence of the scaffold region is engineered guide RNA, characterized in that the following sequences are linked in order from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 10);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. According to claim 1, wherein the sequence of the scaffold region is engineered guide RNA, characterized in that the following sequences are linked in order from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. Engineered guide RNAs for the CRISPR/Cas12f1 system, including:
engineered scaffolds; and
spacer,
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
a first sequence represented by 5'-A-3';
a second sequence represented by 5'-CCGCUUCAC-3' (SEQ ID NO: 432);
a third sequence represented by 5'-UUAG-3';
a fourth sequence represented by 5'-AGUGAAGGUGG-3' (SEQ ID NO: 433);
a fifth sequence represented by 5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
a sixth sequence represented by 5'-AACAAA-3';
linker;
a seventh sequence represented by 5'-GGA-3'; and
The eighth sequence represented by 5'-AUGCAAC-3'. The engineered guide RNA according to claim 5, wherein the linker is 5'-GAAA-3'. 6. The method of claim 5, wherein the linker is 5'-GAAA-3', 5'-UGAAAA-3', 5'-UUGAAAAA-3', 5'-UUCGAAAGAA-3' (SEQ ID NO: 425), 5'-UUCAGAAAUGAA An engineered guide RNA, characterized in that it is selected from the group consisting of -3' (SEQ ID NO: 426), 5'-UUCAUGAAAAUGAA-3' (SEQ ID NO: 427), and 5'-UUCAUUGAAAAAUGAA-3' (SEQ ID NO: 428). 6. The method of claim 5, wherein the sequence of the engineered scaffold region is 5'-A-3', 5'-GA-3', 5'-AGA-3', 5'-GAGA-3', 5'- GGAGA-3', 5'-UGGAGA-3', 5'-GUGGAGA-3', 5'-AGUGGAGA-3', 5'-AAUGGAGA-3', 5'-AAAGUGGAGA-3' (SEQ ID NO: 27), 5'-UAAAAGUGGAGA-3' (SEQ ID NO: 28), 5'-AUAAAGUGGAGA-3' (SEQ ID NO: 29), 5'-GAUAAAAGUGGAGA-3' (SEQ ID NO: 30), 5'-UGAUAAAGUGGAGA-3' (SEQ ID NO: 31) ), 5'-CUGAUAAAAGUGGAGA-3' (SEQ ID NO: 32), 5'-ACUGAUAAAGUGGAGA-3' (SEQ ID NO: 33), 5'-CACUGAUAAAAGUGGAGA-3' (SEQ ID NO: 34), 5'-UCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 34), 5'-UCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 33) 35), 5'-UUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 36), and 5'-CUUCACUGAUAAAGUGGAGA-3' (SEQ ID NO: 37), further comprising a ninth sequence selected from the group consisting of, 3' of the ninth sequence The engineered guide RNA, characterized in that the end is connected to the 5' end of the first sequence. The method of claim 5, wherein the sequence of the engineered scaffold region comprises:
5'-A-3', 5'-AA-3', 5'-AAA-3', 5'-AAAG-3', 5'-AAAGC-3', 5'-AAAGCU-3', 5' consisting of -AAAGCUG-3', 5'-AAAGCUGU-3', 5'-AAAGCUGUC-3', 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), and 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53) a tenth sequence selected from the group, and
5'-U-3', 5'-CU-3', 5'-ACU-3', 5'-AACU-3', 5'-GAACU-3', 5'-AGAACU-3', 5'-UAGAACU-3',5'-UUAGAACU-3',5'-AUUAGAACU-3',5'-GAUUAGAACU-3' (SEQ ID NO: 54), 5'-GGAUUAGAACU-3' (SEQ ID NO: 55), and 5 It additionally comprises an 11th sequence selected from the group consisting of '-GGGAUUAGAACU-3' (SEQ ID NO: 56),
The 3' end of the second sequence and the 5' end of the third sequence are linked through the 10th sequence,
The engineered guide RNA, characterized in that the 3' end of the third sequence and the 5' end of the fourth sequence are linked through the eleventh sequence. 10. The method of claim 9,
When the tenth sequence is 5'-A-3', the eleventh sequence is 5'-U-3',
When the tenth sequence is 5'-AA-3', the eleventh sequence is 5'-CU-3',
When the tenth sequence is 5'-AAA-3', the eleventh sequence is 5'-ACU-3',
When the tenth sequence is 5'-AAAG-3', the eleventh sequence is 5'-AACU-3',
When the tenth sequence is 5'-AAAGC-3', the eleventh sequence is 5'-GAACU-3',
When the tenth sequence is 5'-AAAGCU-3', the eleventh sequence is 5'-AGAACU-3',
When the tenth sequence is 5'-AAAGCUG-3', the eleventh sequence is 5'-UAGAACU-3' or 5'-UUAGAACU-3',
When the tenth sequence is 5'-AAAGCUGU-3', the eleventh sequence is 5'-AUUAGAACU-3',
When the tenth sequence is 5'-AAAGCUGUC-3', the eleventh sequence is 5'-GAUUAGAACU-3' (SEQ ID NO: 54),
When the tenth sequence is 5'-AAAGCUGUCC-3' (SEQ ID NO: 52), the eleventh sequence is 5'-GGAUUAGAACU-3' (SEQ ID NO: 55),
When the tenth sequence is 5'-AAAGCUGUCCC-3' (SEQ ID NO: 53), the eleventh sequence is 5'-GGGAUUAGAACU-3' (SEQ ID NO: 56),
When the tenth sequence is 5'-AAAAGCUGUCCC-3' (SEQ ID NO: 440), the eleventh sequence may be 5'-GGGAUUAGAACUU-3' (SEQ ID NO: 442).
Where the tenth sequence is 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441), the eleventh sequence is 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443). 11. The engineered guide according to claim 10, wherein the tenth sequence is 5'-CAAAAGCUGUCCC-3' (SEQ ID NO: 441) and the eleventh sequence is 5'-GGGAUUAGAACUUG-3' (SEQ ID NO: 443). RNA. The method of claim 5, wherein the sequence of the engineered scaffold region comprises:
5'-U-3', 5'-UU-3', 5'-UUC-3', 5'-UUCA-3', 5'-UUCAU-3', 5'-UUCAUU-3', and 5 12th sequence selected from the group consisting of '-UUCAUUU-3', and
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-UGAA-3', 5'-AUGAA-3', 5'-AAUGAA-3', and 5 It additionally includes a 13th sequence selected from the group consisting of '-GAAUGAA-3',
In this case, the 3' end of the sixth sequence and the 5' end of the linker are connected through the 12th sequence,
The engineered guide RNA, characterized in that the 3' end of the linker and the 5' end of the 7th sequence are linked through the 13th sequence. 13. The method of claim 12,
when the twelfth sequence is 5'-U-3', the thirteenth sequence is 5'-A-3',
when the twelfth sequence is 5'-UU-3', the thirteenth sequence is 5'-AA-3',
when the twelfth sequence is 5'-UUC-3', the thirteenth sequence is 5'-GAA-3',
when the twelfth sequence is 5'-UUCA-3', the thirteenth sequence is 5'-UGAA-3',
When the twelfth sequence is 5'-UUCAU-3', the thirteenth sequence is 5'-AUGAA-3',
when the twelfth sequence is 5'-UUCAUU-3', the thirteenth sequence is 5'-AAUGAA-3',
When the twelfth sequence is 5'-UUCAUUU-3', the thirteenth sequence is 5'-GAAUGAA-3'. Engineered guide RNAs for the CRISPR/Cas12f1 system, including:
engineered scaffold area; and
spacer;
In this case, the spacer has a length of 10 to 50 nucleotides, and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region comprises the following sequence:
5' end to 3' end direction,
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), a sequence selected from the group consisting of 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9),
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order,
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11), and
5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAAUUCA-3' (SEQ ID NO: 66), 5'-AACAAAUUCAU- an engineered tracrRNA to which a sequence selected from the group consisting of 3' (SEQ ID NO: 67), 5'-AACAAAUUCAUU-3' (SEQ ID NO: 68), and 5'-AACAAAUUCAUUU-3' (SEQ ID NO: 12) is linked; and
5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3', 5'-AUGAAGGA-3', 5'-AAUGAAGGA-3', and a sequence selected from the group consisting of 5'-GAAUGAAGGA-3' (SEQ ID NO: 14), and
5'-AUGCAAC-3' linked engineered crRNA repeat sequence portion,
At this time, the 3' end of the engineered crRNA repeat sequence portion is connected to the 5' end of the spacer,
If the sequence of the engineered tracrRNA is identical to 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 1), except that the sequence is identical to that of the engineered tracrRNA repeats that the sequence is identical to 5'-CUUCACUGAUAAAGUGGAGAGAACCGCUUCACCAAAAGCUGUCCCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (SEQ ID NO: 1). characterized. An engineered CRISPR/Cas12f1 complex capable of editing a nucleic acid comprising a target sequence, comprising:
Cas12f1 protein; and
engineered guide RNA,
In this case, the engineered guide RNA comprises:
engineered scaffold area; and
spacer;
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and
5'-AUGCAAC-3'. The engineered CRISPR/Cas12f1 complex according to claim 15, wherein the sequence of the scaffold region included in the engineered guide RNA is sequentially linked from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 10);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. The engineered CRISPR/Cas12f1 complex according to claim 15, wherein the sequence of the scaffold region included in the engineered guide RNA is sequentially linked from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. An engineered CRISPR/Cas12f1 complex capable of editing a nucleic acid comprising a target sequence, comprising:
Cas12f1 protein; and
An engineered guide RNA according to any one of claims 5 to 13. A vector capable of expressing each component of the CRISPR/Cas12f1 system, comprising:
a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein;
a first promoter sequence operably linked to said first sequence;
a second sequence comprising a nucleic acid sequence encoding the engineered guide RNA; and
a second promoter sequence operably linked to said second sequence;
In this case, the engineered guide RNA comprises:
engineered scaffold area; and
spacer;
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and
5'-AUGCAAC-3'. 20. The vector according to claim 19, wherein the sequence of the scaffold region included in the engineered guide RNA is sequentially linked from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 10);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. 20. The vector according to claim 19, wherein the sequence of the scaffold region included in the engineered guide RNA is sequentially linked from the 5' end to the 3' end:
5'-A-3';
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430);
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110); and
5'-AUGCAAC-3'. The vector according to claim 19, wherein the vector is at least one selected from the group consisting of a plasmid, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus. A vector capable of expressing each component of the CRISPR/Cas12f1 system, comprising:
a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein;
a first promoter sequence operably linked to said first sequence;
a second sequence comprising a nucleic acid sequence encoding the engineered guide RNA according to any one of claims 5 to 13; and
a second promoter sequence operably linked to said second sequence. A method of editing a nucleic acid comprising a target sequence in a cell, comprising:
delivering the Cas12f1 protein or a nucleic acid encoding the same, and an engineered guide RNA or a nucleic acid encoding the name into a cell;
Due to this, the CRISPR/Cas12f1 complex may be formed in the cell,
Due to this, the nucleic acid comprising the target sequence can be edited by the CRISPR/Cas12f1 complex,
The engineered guide RNA is characterized in that it comprises:
engineered scaffold area; and
spacer;
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is characterized in that it differs from 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 7),
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), and 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) a sequence selected from SEQ ID NO: 117); and
5'-AUGCAAC-3'. A method of editing a nucleic acid comprising a target sequence in a cell, comprising:
delivering the Cas12f1 protein or a nucleic acid encoding the same, and an engineered guide RNA or a nucleic acid encoding the name into a cell;
Due to this, the CRISPR/Cas12f1 complex may be formed in the cell,
Due to this, the nucleic acid comprising the target sequence can be edited by the CRISPR/Cas12f1 complex,
The engineered guide RNA is characterized in that it comprises:
engineered scaffold area; and
spacer; and
At this time, in the direction from the 5' end to the 3' end of the engineered guide RNA, the engineered scaffold region, and the spacer are connected in order,
The spacer comprises 10 or more and 50 or less nucleotides and has a sequence complementary to the target sequence,
The sequence of the engineered scaffold region is 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUUCCUCUCCCAAAAUUCGAGAACCAAUGAAUGAAUUCGAUGAAGAAUGAAUGAAUGAUGAAGUGAGAAUGAAUGAAUGAUGAUGAGAAUGAAUGAAUGAUGAAUGAAGAAU characterized by that the sequence differs by the sequence
The sequence of the engineered scaffold region consists of the following sequences linked in order from the 5' end to the 3' end:
5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5'-GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3',5'-GUGGAGAA-3',5'-AGUGGAGAA-3',5'-AAGUGGAGAA-3' (SEQ ID NO: 16), 5'-AAAGUGGAGAA-3' (SEQ ID NO: 17), 5'-UAAAGUGGAGAA-3' (SEQ ID NO: 18), 5'-AUAAAGUGGAGAA-3' (SEQ ID NO: 19), 5'-GAUAAAGUGGAGAA-3' (SEQ ID NO: 20), 5'-UGAUAAAGUGGAGAA-3' (SEQ ID NO: 21), 5'-CUGAUAAAGUGGAGAA-3' (SEQ ID NO: 22), 5'-ACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 23), 5'-CACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24), 5'-UCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 25 ), 5'-UUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 26), and 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 9);
5'-CCGCUUCACUUAGAGUGAAGGUGG-3' (SEQ ID NO: 430), 5'-CCGCUUCACCUUAGGAGUGAAGGUGG-3' (SEQ ID NO: 431), 5'-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3' (SEQ ID NO: 38), 5'-CCGCUUCACCAAUUAGUUGAGUGAAGGG-3' (SEQ ID NO: 39) ), 5'-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3' (SEQ ID NO: 40), 5'-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3' (SEQ ID NO: 41), 5'-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGCUAAGUGG-3' (SEQ ID NO: 42), 5'-AGAGGUCCG'UGAAGGUGG-3' (SEQ ID NO: 42), 5'-AGAG No. 43), 5'-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 44), 5'-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 45), 5'-CCGCUUAUCACCAAAAGCUGUGUUAGUAAGAAGGUAGGUAGGUAAGAUAAGAUGAGUUAGUAAGUAACUUGACUGAGUAGGUA-3' (SEQ ID NO: 47), 5'-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 48), 5'-CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG-3' (SEQ ID NO: 49), and 5'-GUCCUGAGUUCACCAAAAGGAUGGAUGA selected from the group consisting of: order;
5'-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA-3' (SEQ ID NO: 11);
5'-AACAAAGAAAGGA-3' (SEQ ID NO: 110), 5'-AACAAAUGAAAAGGA-3' (SEQ ID NO: 111), 5'-AACAAAUUGAAAAAGGA-3' (SEQ ID NO: 112), 5'-AACAAAUUCGAAAGAAGGA-3' (SEQ ID NO: 113) ), 5'-AACAAAUUCAGAAAUGAAGGA-3' (SEQ ID NO: 114), 5'-AACAAAUUCAUGAAAAUGAAGGA-3' (SEQ ID NO: 115), 5'-AACAAAUUCAUUGAAAAAUGAAGGA-3' (SEQ ID NO: 116), 5'-AACAAAUUCAUUUGAAAGAAUGAAGGA-3' (SEQ ID NO: 115) No. 117), 5'-AACAAAUUCAUUUUGAAACGAAUGAAGGA-3' (SEQ ID NO: 293), 5'-AACAAAUUCAUUUUUGAAAACGAAUGAAGGA-3' (SEQ ID NO: 294), 5'-AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAACGAAUGAAGGAUUGAAUGAAU-GAAAAAGGAUGA (SEQ ID NO: 296), 5'-AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 297), 5'-AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 298), 5'-AACAAAUUCAUUCAUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAUGAAUUCAUUUUUUCCUACAACUGAAUGAAUUUUUCCUCUGAAUGAAGAAUUCA 3' (SEQ ID NO: 300), 5'-AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 301), 5'-AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAGU-3' (SEQ ID NO: 302), 5'-AACAAAUCUCCAUGAAUGAU AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 304), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 305), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 306), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 307), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 308 ), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 309), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 310), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열번호 311), 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3'(서열 312), 5'-AACAAAUUCAUUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 313), and 5'-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO:314) selected from the group consisting of; and
5'-AUGCAAC-3'. 26. The method of any one of claims 24 or 25, wherein said delivery is intracellular injection of said Cas12f1 protein and said engineered guide RNA as a CRISPR/Cas12f1 complex. 25. The method of claim 24, wherein said delivering is intracellular injection of a vector comprising a nucleic acid encoding said Cas12f1 protein and a nucleic acid encoding said engineered guide RNA. 26. The method of any one of claims 24 or 25, wherein the cell is a eukaryotic cell. The method of claim 27, wherein the vector is one or more selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, adeno-associated virus, vacciniavirus, poxvirus and herpes simplex virus. A method of editing a nucleic acid comprising a target sequence in a cell, comprising:
Delivering the Cas12f1 protein or a nucleic acid encoding the same, and the engineered guide RNA according to any one of claims 5 to 13 or a nucleic acid encoding the name into a cell,
Due to this, the CRISPR/Cas12f1 complex may be formed in the cell,
This allows the nucleic acid containing the target sequence to be edited by the CRISPR/Cas12f1 complex. A DNA encoding the engineered guide RNA of any one of claims 1 or 2. A DNA encoding the engineered guide RNA of any one of claims 5-13. A DNA encoding the engineered guide RNA of claim 14 .

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