KR20160133380A - Targeted genome editing based on CRISPR/Cas9 system using short linearized double-stranded DNA - Google Patents

Abstract

The present invention relates to a target genome editing using a CRISPR-Cas9 system using linear double-stranded DNA. More specifically, the linear double-stranded DNA in which guide RNA and donor DNA are bonded together is used in CRISPR-Cas9-based genome editing, thereby inducing substitution mutation in eukaryotic or procaryotic cells with high efficiency.

Description

{Targeted genome editing based on CRISPR / Cas9 system using short linearized double-stranded DNA}

The present invention relates to a target genetic correction using a CRISPR / Cas9 system utilizing linear double stranded DNA expressing guide RNA and donor DNA.

There are many potential causal variants associated with Mendelian diseases and somatic variants associated with cancer from next-generation sequencing (NGS) and microarray-based gene studies, Thousands of gene loci related to human traits have been identified.

Genome editing based on the recently developed RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats) -related nuclease Cas9 has been used for target knock-out, transcriptional activation and single guide RNA (sgRNA ) (I. E., A crRNA-tracrRNA fusion transcript), which demonstrates extensibility by targeting a number of gene locations. However, unlike knock-out or transcriptional regulation, the occurrence of CRISPR-based single nucleotide variation (SNV) is largely limited to a single genomic locus. Recently, an oligonucleotide-mediated homologous recombination (HR) strategy, a widely known strategy for E. coli engineering, has been proposed to introduce multisubstitution mutations in human cells, but the rare occurrence of genome-mathematical events (10 ⁵ to 10 ⁷ One of the cells) and low yields.

 M. Jinek et al. Science 2012, 337, 816-821  O. Shalem et al. Science 2014, 343, 84-87  T. Wang et al. Science 2014, 343, 80-84  L. A. Gilbert et al. Cells 2014, 159, 647-661  S. Konermann et al. Nature 2015, 517, 583-588  L. Cong et al. Science 2013, 339, 819-823  P. Mali et al. Science (New York, N.Y.) 2013, 339, 823-826  D. Yu et al. Proceedings of the National Academy of Sciences of the United States of America 2000, 97, 5978-5983  H. H. Wang et al. Nature 2009, 460, 894-898

It is an object of the present invention to provide a linear double-stranded DNA which induces a substitution mutation in a highly efficient manner by transferring a form in which a guide RNA and a donor DNA are coupled to a cell in a CRISPR-Cas9-based dielectric correction system.

It is another object of the present invention to provide a CRISPR-Cas9-based dielectric calibration use of said linear double-stranded DNA.

In order to accomplish the above object, the present invention provides a promoter for expressing a guide RNA; DNA encoding guide RNA; Terminator; And donor DNA. ≪ Desc / Clms Page number 2 >

The present invention also relates to said linear double stranded DNA; And a vector expressing the Cas9 protein or the Cas9 protein. The present invention provides a CRISPR-Cas9-based dielectric correcting composition.

The present invention provides the effect of inducing high-efficiency substitution mutations in eukaryotic cells or prokaryotic cells by using linear double-stranded DNA in which guide RNA and donor DNA are combined for CRISPR-Cas9-based genome correction.

Figure 1 is a diagram illustrating the need for a delivery system via donor DNA binding with the sgRNA of the present invention.
Figure 2 shows the usefulness of the sgR-DNA delivery method of the present invention compared to the conventional delivery method.
Figure 3 shows the sgR-DNA construct of the present invention.
FIG. 4 shows a process of synthesizing the sgR-DNA construct of the present invention for delivery to E. coli.
FIG. 5 shows a process of synthesizing the sgR-DNA construct of the present invention for delivery to human cells.
FIG. 6 shows an experimental procedure for calibrating the sgR-DNA construct of the present invention and galK gene synthesized for delivery to E. coli.
FIG. 7 shows a process for synthesizing linear double-stranded DNA expressing sgRNA for delivery to E. coli.
8 shows the homologous recombination efficiency at the position of E. coli galK according to the donor length (63 nt, 93 nt, 123 nt).
Fig. 9 shows the homologous recombination efficiency when sgRNA and ssODN and sgR-DNA were used at the position of E. coli galK.
FIG. 10 shows the results of experiments using eleven aro genes distributed throughout the target dielectric and an sgR-DNA library targeting the same for confirmation of the multi-position dielectric correction of E. coli.
Figure 11 shows the multi-site dielectric correcting efficiency of E. coli.
Figure 12 shows the sgR-DNA construct of the present invention synthesized for delivery to human cells.
Figure 13 shows donor DNA with four mismatched sequences for the EGFR target.
Fig. 14 shows a process for synthesizing linear double-stranded DNA expressing sgRNA for delivery to human cells.
Fig. 15 shows homologous recombination efficiency at the human cell EGFR site.
Figure 16 shows the insertion-deletion efficiency at the human cell EGFR position.
Figure 17 shows the genotype of the target gene position in a single clone through Sanger sequencing.
Figure 18 is a graph showing that the insert-deficiency efficiency at the off-target position is lower than the EGFR target position.
Figure 19 shows the ratio distribution of insertion-defect occurrences for each base position at the off-target position.
Figure 20 shows the distribution of insertion-deficit sizes at off-target locations.
21 shows the substitution efficiency at the BRAF position.
22 shows the substitution efficiency at the KRAS position.
FIG. 23 shows experiments using 10 genes distributed throughout the genome and the sgR-DNA library targeted thereto for confirmation of the multi-positional genetic correction of human cells.
Figure 24 shows the multimode dielectric calibration efficiency of human cells.
25 shows a process of constructing an sgR-DNA library using a cleaved oligonucleotide pool from a programmable microarray.

In CRISPR / Cas9-based dielectric calibrations, we hypothesize that the lack of high-efficiency replacement mutagenesis techniques may result from the absence of a suitable delivery system. Unlike the knock-out formula using a sgRNA vector as a single target-specific component, the substitution formula requires an additional donor DNA comprising the designed mutation, so to position the sgRNA at the target site and the matching donor DNA, (Fig. 1). Based on this hypothesis, the present inventors have found that by assembling DNA and donor DNA encoding an sgRNA transcript which can be prepared more easily than prior transcription methods to facilitate CRISPR / Cas9-based displacement correction in a multiple position calibration scheme, DNA (i.e., matched sgRNA-donor DNA pair) was developed (Fig. 2). To verify the capabilities of the CRISPR / Cas9-based system of the present invention, this system was applied to E. coli engineering and then expanded to mammalian cell engineering. The present invention was then completed by observing whether the sgR-DNAs pool could be used for multiple generation of substitutional mutations in both E. coli (11-plex) and the human genome (10-plex).

Thus, the invention provides a promoter for expressing a guide RNA; DNA encoding guide RNA; Terminator; And donor DNA. ≪ Desc / Clms Page number 2 >

The present invention also relates to said linear double stranded DNA; And a vector expressing the Cas9 protein or the Cas9 protein. The present invention provides a CRISPR-Cas9-based dielectric correcting composition.

The linear double-stranded DNA of the present invention is a structure capable of expressing a guide RNA that recognizes a target gene in a CRISPR-Cas9 system, and has a structure in which a DNA encoding a guide RNA and a donor DNA are combined, The DNA pair is simultaneously transferred into the cell, and when the target DNA is cleaved by the Cas9 protein, the donor DNA including the mismatch codon is included in the genome in place of the target DNA through the homologous recombination of the donor DNA to increase the substitution mutation efficiency of the target DNA .

As used herein, the term "truncation" means the breakage of the covalent backbone of a nucleotide molecule.

Preferably, the linear double-stranded DNA of the present invention comprises a promoter for expressing the guide RNA; DNA encoding guide RNA; Terminator; And the donor DNA may be sequentially connected. Therefore, the linear double-stranded DNA transferred into the cell is transcribed by the promoter into the guide RNA, and the transcription is terminated through the terminator, so that the transcription of the donor DNA does not occur. The donor DNA participates in homologous recombination after cleavage of the target DNA to induce substitution mutations of the target DNA.

The target DNA may be an endogenous DNA or an artificial DNA, but is preferably an intrinsic DNA.

Since the linear double-stranded DNA can be easily produced in a microarray, the production process is simple compared to the prior art using a vector, the cost is reduced, and the DNA can be used for the genetic correction of prokaryotic or eukaryotic cells, .

The promoter is a promoter for expressing a guide RNA specific for a target DNA in a cell, and can be appropriately adopted at the level of those skilled in the art depending on the kind of cell, and is not particularly limited. For example, T7 promoter, SP6 promoter, rpr-1 Promoters, rrk promoters, or promoters that operate on the genome of the organism to be corrected, such as the U6 promoter, can be used.

The guide RNA is RNA specific to the target DNA, which is expressed through transcription of linear double-stranded DNA transferred into the cell, recognizes the target gene, forms a complex with the Cas9 protein, and brings the Cas9 protein into the target DNA . Thus, the guide RNA may comprise a spacer capable of recognizing the target DNA; And a guide RNA scaffold consisting of a non-variable sequence that is independent of the target.

The spacer refers to a sequence that allows the guide RNA to recognize the target DNA, and may include some or all of the protospacer adjacent motifs (PAMs) sequences near the target DNA location. Preferably, sequences adjacent to the PAMs can be used. In addition, the spacer may be suitably modified depending on the type of the target cell. According to one embodiment of the present invention, a spacer can be prepared by joining Extra 5'G to a sequence adjacent to PAM for introduction into human cells.

The guide RNA scaffold may consist of two RNAs, namely, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or may be a single chain RNA produced by fusion of an essential part of crRNA and tracrRNA (single-chain guide RNA, sgRNA). The guide RNA may be a dual RNA including crRNA and tracrRNA. Any guide RNA scaffold can be used in the present invention, provided that the guide RNA scaffold comprises an essential portion of the crRNA and the tracrRNA and a portion complementary to the target. The crRNA may be hybridized with the target DNA.

Preferably, it may be a single-chain guide RNA.

The terminator is connected to the DNA terminus coding for the guide RNA to terminate the transcription of the DNA encoding the guide RNA. The terminator is not particularly limited and can be used at a suitable selection level by those skilled in the art according to the promoter. For example, RNA polymerase III terminator or -TTTTTT- sequence.

The donor DNA may comprise a PAM sequence and consist of a homologous sequence comprising a mutated codon having a mismatch of 1 to 3 nucleotides with respect to the target DNA.

In addition, the donor DNA may be designed to include PAM having the same codon after substitution so that the PAM sequence is included in the donor DNA sequence so that no further recognition of the PAM sequence occurs after the base mutation occurs.

The Cas9 protein means an essential protein element in the CRISPR / Cas9 system and when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), the active endonuclease . Information on Cas9 genes and proteins is available from, but is not limited to, GenBank in the National Center for Biotechnology Information (NCBI).

When the Cas9 protein is delivered into a cell, it may be linked to a protein transduction domain. The protein transfer domain may be a poly-arginine domain or a TAT protein derived from HIV, but is not limited thereto.

The Cas9 protein may be delivered via transfection in the form of a vector containing Cas9 protein, that is, a vector comprising Cas9 protein coding nucleic acid. The Cas9 protein coding nucleic acid may be in the form of a vector, such as a plasmid, containing a Cas9 coding sequence under a promoter such as CMV or CAG.

The delivery of the linear double-stranded DNA and the Cas9 protein or a vector expressing the Cas9 protein can be performed by microinjection, electroporation, DEAE-dextran treatment, lipofection, But are not limited to, particle-mediated transfection, protein transfer domain mediated introduction, virus-mediated gene delivery, and PEG-mediated transfection in protozoa.

Preferably, the vector expressing the linear double-stranded DNA and Cas9 protein or Cas9 protein can be delivered to prokaryotic or eukaryotic cells via co-transfection or serial-transfection .

The stepwise transfection may be performed by transfecting a prokaryotic or eukaryotic cell with a vector expressing Cas9 protein or Cas9 protein, and then transfecting linear double stranded DNA.

The CRISPR-Cas9 system consisting of the linear double-stranded DNA of the present invention and the Cas9 protein can be useful for inducing targeted mutations of a single site or multiple locus genes in prokaryotic or eukaryotic cells.

The eukaryotic cell or the prokaryotic cell may be a cell such as Escherichia coli, yeast, fungus, plant, insect, amphibian, mammal and the like, and may be, for example, a cell cultured in in vitro commonly used in the art, But are not limited to, primary cell cultures, in vivo cells, and cells of mammals including humans.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Linear Double strand sgR -DNA design

(T7 polymerase, Cas9 , sgRNA  And ssODNs  Expressing plasmid)

Plasmid N249 encoding T7 polymerase was obtained from George Church (Harvard Medical School) and pET-Cas9 encoding Cas9 was constructed by cloning Cas9 sequence from pMJ806 (Addgene plasmid 39312) into pET-28a. Plasmid (Addgene plasmid 44719) expressing the GFP-linked human codon-optimized Cas9 nuclease and gRNA (Addgene plasmid 41824) under the control of the U6 promoter for transcription were purchased from Addgene (USA). Double stranded DNA containing the target site was cloned into an AflII-digested gRNA cloning vector. The ssODNs for the target gene positions were synthesized in IDT (USA). Single-stranded oligodeoxynucleotide (ssODNs) sequences for E. coli and human cells are shown in Table 1.

( sgR- DNA library design)

The sgR-DNA construct contains the T7 promoter (24 bp) or the U6 promoter (264 bp), the DNA encoding the sgRNA (97 bp), the terminator sequence (6 Ts) and the donor DNA (63 bp, 93 bp, (Fig. 3).

First, for sgRNA design, protospacer adjacent motifs (PAMs) near the target gene location were selected, and a sequence close to the selected PAM was determined as a spacer of the sgRNA (SEQ ID NO. 1-12). In sgR-DNA for human cells, a spacer of sgRNA was made by attaching an extra 5'G to the 19-bp sequence close to PAM. A 20-bp spacer was fused to the 77-bp scaffold to make a DNA encoding sgRNA (SEQ ID NO. 13-25).

Donor DNA was constructed to include the 3-nt mutation codon and 30-nt, 45-nt or 60-nt homology arms (SEQ ID NO. 26-52). It was also designed to introduce mutated codons with at least 2-nt mismatches along with the target codon, allowing them to be distinguished from NGS errors. In addition, the donor DNA was designed to include PAM with the same codon after substitution so that no further recognition of the PAM sequence occurred after the SNV event. Subsequently, sgR-DNA was constructed through assembly of DNA and donor DNA encoding the sgRNA.

(For delivery to E. coli sgR -DNA library Construct )

The sgR-DNA constructs (190 bp, 220 bp or 250 bp containing 30-nt, 45-nt or 60-nt homologous arms, respectively) were synthesized in three parts:

i) a sequence (20 nt) encoding the right portion of the T7 promoter (20 nt), the spacer (20 nt) and the region corresponding to the sgRNA scaffold;

ii) sequence (77 nt) encoding the sgRNA scaffold, terminator sequence (6Ts); And

iii) Donor DNA (63 nt, 93 nt, or 123 nt) (Figure 4).

Each part was independently synthesized by solid-state oligonucleotide synthesis (IDT, USA).

i), ii) and iii) were assembled using T7 forward primer, target specific middle oligo (sequence shown in Table 1).

For single position correction, the composition for the synthesis of sgR-DNA from oligonucleotides is as follows: 1 [mu] M T7_fwd_primer (SEQ ID NO. 53) 1 [mu] M galK_gRNA (SEQ ID NO. 54) 1 μl of 1 μM galK_middle (SEQ ID NO: 56), 1 μl of 10 μM galK_63_donor_rev or galK_93_donor_rev or galK_123_donor_rev (SEQ ID NO: 26-28) 10 mu l, distilled water 5 mu l. To correct for the 11 position of the E. coli genome (gene aroA, aroB, aroC, aroD, aroE, aroF, aroG, aroK, aroL, aroM, aroP), oligonucleotide compositions for synthesizing sgR-DNA from nucleotides are as follows: :

1 μM aroX_gRNA (SEQ ID NO: 57-67), 1 μM gRNA_rev (SEQ ID NO: 55) 1 μl, 1 μM aroX_middle (SEQ ID NO: 68-78 1 μl, 10 μM aroX_donor_rev (SEQ ID NO. 29-39) 1 μl, 2 μl Pfu polymerase master mix (intron) 10 μl, distilled water 5 μl

PCR was performed with i-pfu 2 x PCR Master Mix Solution (Intron, Korea) to make sgR-DNA construct, and PCR cycling was performed as follows: initial denaturation at 95 ° C for 5 minutes; Subsequently, 20 cycles of denaturation at 95 DEG C for 30 seconds, annealing at 50 DEG C for 30 seconds, and extension at 72 DEG C for 30 seconds; And final extension at 72 ° C for 5 minutes.

The assembled construct was separated from 2% agarose gel and purified on a MinElute Gel Extraction Kit (Qiagen, Germany).

(For delivery to human cells) sgR -DNA library Construct )

The sgR-DNA construct (490 bp) was synthesized in three parts:

i) the U6 promoter (264 bp);

ii) a sequence (19 nt) encoding the region corresponding to the U6 promoter, a spacer (20 nt), a sequence encoding 77 nt of the sgRNA scaffold, a terminator sequence (6Ts) 35 nt); And

iii) Donor DNA (123 nt) (Figure 5).

Each part was synthesized independently. The i) portion was amplified from the gRNA cloning vector using the U6 fwd primer (SEQ ID NO. 79) and the rev primer (SEQ ID NO. 80) for human cell application to generate the double stranded U6 promoter (SEQ ID NO. 81 ) (SEQ ID NOs: 82-94) and iii) (SEQ ID NOs: 40-52) were obtained in solid-phase oligonucleotide synthesis (IDT, USA) (Table 1), and these portions were assembled using PCR.

The sgR-DNA construct was constructed by PCR with i-pfu 2x PCR Master Mix Solution (Intron, Korea). The composition for the synthesis of the respective sgR-DNA from the double-stranded U6 promoter and the oligonucleotide is as follows: 1 [mu] M U6_fwd_primer (SEQ ID NO: 79), 1 [mu] M double- stranded U6 promoter 1 [ 1 [mu] M gene_gRNA (SEQ ID NO: 82-94), 1 [mu] M gene_donor_rev (SEQ ID NO: 40-52), 10 [mu] Pfu polymerase master mix (intron) The mixture of the composition was amplified through the following PCR reactions: initial denaturation at 95 ° C for 5 minutes; Subsequently, 20 cycles of denaturation at 95 DEG C for 30 seconds, annealing at 58 DEG C for 30 seconds, and extension at 72 DEG C for 30 seconds; And final extension at 72 ° C for 5 minutes.

The assembled construct was separated from 2% agarose gel and purified on a MinElute Gel Extraction Kit (Qiagen, Germany).

(E. coli cell culture and electroporation)

E. coli strain EcHB3 (EcNR2 derivative;. Inactivation of cat, bla, galK, malK by MAGE, the sequences of targeting oligonucleotides are from Wang et al) obtains from HB Kim, appropriate antibiotics (Kanamycin, 30 ㎍ / mL; and spectinomycin , 100 [mu] g / mL) was added at 30 [deg.] C in LB or LB agar plates. The lac promoter was induced using 1 mM IPTG.

Electroporation was performed using a Bio-Rad Gene Pulser. When pN249 or pET-Cas9 plasmids were electroporated, cells were incubated at 30 ° C until OD ₆₀₀ = ~ 0.8. 1 mL of cells were harvested by centrifugation at 15000 rpm for 1 minute. The pellet was washed twice with 1 mL of cold ddH ₂ O and resuspended with 50 μl of the appropriate plasmid. In a 1-mm gap cuvette, the cell supernatant was electroporated with a pulse of 1.8 kV, added to LB medium without antibiotics, restored for 3 hours and transferred to LB supplemented with appropriate antibiotics.

For electroporation of sgR-DNA or linear DNA fragments, cells were grown in the presence of 1 mM IPTG to express Cas9 or T7 polymerase modulated by the IPTG inducible promoter (pT7lacO promoter). The cells were heat-shocked at mid-exponential (OD ₆₀₀ = ~ 0.8) for 15 min at 42 ° C to allow expression of the λ-red recombination system regulated by a heat-inducible promoter (pL promoter). Then, 1 mL of cells were harvested, washed twice with ddH ₂ O, and then pulsed with 0.1 μM or 1 μM of linear DNA fragments. To confirm the manipulation efficiency, electroporation was performed to amplify the target region, and the cells were incubated overnight.

(Human cell culture and Transfection )

(5%) in Dulbecco's modified Eagle medium (Gibco / Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco / Life Technologies) and 1% penicillin / streptomycin HEK 293T cells were cultured under the humidifying condition of CO ₂ . On the day before transfection, cells were plated in 6-well plates or 100-mm culture dishes.

For single-site calibration experiments, 1.6 μg of sgR-DNA and / or 0.8 μg of pCas9-GFP were transfected into cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions.

For multi-site calibration experiments, 10 ug of sgR-DNA and 5 ug of pCas9-GFP were transfected into the cells.

( Flow Saitometry  analysis)

HEK 293T cells were selected using BD-FACS Aria II or III instrument (BD Biosciences, USA) 48 hours after transfection with pCas9-GFP and sgR-DNA library. Briefly, cultured cells were trypsinized and centrifuged to obtain pellets and washed with PBS. The pellet is resuspended to a (PBS containing 2mM EDTA, 25mM HEPES, and 1% bovine serum albumin) sorting buffer, and the final density to be 3-4 × 10 ⁶ cells / mL. Finally, cells were filtered through a cell strainer to prepare a single-cell suspension.

(Clonal proliferation and genotype analysis)

GFP-positive cells transfected with pCas9-GFP and sgR-DNA at 48 hours post-transfection were seeded into wells of a 96-well plate containing complete medium to form clones with defined mutations Plated. Cells were grown for 2-3 weeks and then genomic DNA was extracted from the clonal cell line. The target region was amplified by PCR and genotyped using Sanger sequencing.

(Separation of genomic DNA)

Genomic DNA was isolated after transfection or 48 hours after FACS analysis using DNeasy Blood and Tissue Kit (Qiagen, Germany). The cells were harvested, the cell pellet resuspended in 200 [mu] l of PBS and lysed with proteinase K. [ The cell lysate was loaded onto a spin column, washed twice and eluted with nuclease-free water. The isolated genomic DNA was quantitated using Qubit dsDNA BR Assay Kit (Life Technologies, USA).

(Confirmation of target expression by sequencing in E. coli experiment)

The PCR primers were designed to amplify the target region where the size of the PCR product was 200bp in mass (Table 1). To prevent capture of the donor DNA, the primer was designed to capture a dielectric region sufficiently far away from the codon made for the modification.

The PCR composition for PCR amplification of the galK gene in the E. coli genome was as follows: 1 μl of E. coli, 1 μl of 10 μM galK_fwd_primer (SEQ ID NO. 95), 1 μl of 10 μM galK_rev_primer (SEQ ID NO. 96), 2 × KAPA HiFi 10 μl of HotStart ReadyMix, 3 μl of distilled water

PCR was amplified using the respective aroX_fwd_primer and aroX_rev_primer pairs (SEQ ID NO: 41-62) for 11 positions in the E. coli genome. The PCR composition was as follows: Escherichia coli 5 μl, 10 μM aroX_fwd_primer (SEQ 1 μl, 10 μM aroX_rev_primer (SEQ ID NO: 108-118) 1 μl, 2 × KAPA HiFi HotStart ReadyMix 10 μl, distilled water 3 μl

The target region was amplified using Kapa HiFi HotStart ReadyMix (Kapa Biosystems, USA) under the following cycling conditions: initial denaturation at 95 ° C for 3 minutes; Subsequently, denaturation at 95 DEG C for 20 seconds, annealing at 60 DEG C for 15 seconds, and extension at 72 DEG C for 15 seconds were 27 cycles; And final extension at 72 ° C for 1 minute.

PCR products were collected and prepared with SPARK DNA Sample Prep Kit (Enzymatics, USA). Finally, genome sequence information was obtained for 11 sites by next sequencing using HiSeq 2500 system (Illumina, USA).

(Confirmation of target expression by sequencing in human cell experiments)

In a single position calibration experiment, a PCR primer was designed to amplify a target region of approximately 400 bp in size of the PCR product (Table 1).

In a multiple position calibration experiment, the capture region starts at 100-134 bp upstream of the substitution site and reaches 100-129 bp downstream (Table 1). To prevent capture of the donor DNA, the primer was designed to capture a dielectric region sufficiently far away from the codon made for the modification.

PCR amplification was carried out using the respective primer pairs (SEQ ID NO. 119-124) only for the corresponding gene part in which the mutation was introduced for single position calibration confirmation. The PCR composition was as follows: 1 μl of 200 ng of genomic DNA, 10 μM 1 [mu] l of 10 [mu] M gene_rev_primer (SEQ ID NO: 122-124), 2 [mu] KAPA HiFi HotStart ReadyMix 10 [mu] l, distilled water 3 [

PCR amplification was performed using the respective primer pairs (SEQ ID NO: 125-144) corresponding to the 10 positions at which the mutations were introduced for confirmation of the multiple position. The PCR composition was as follows: 1 μl of 200 ng of genomic DNA , 1 μl of 10 μM gene_fwd_primer (SEQ ID NO. 125-134), 10 μM gene_rev_primer (SEQ ID NO: 135-144), 2 μl KAPA HiFi HotStart ReadyMix 10 μl, distilled water 3 μl

The target region was amplified using Kapa HiFi HotStart ReadyMix (Kapa Biosystems, USA) under the following cycling conditions: initial denaturation at 95 ° C for 3 minutes; Subsequently, denaturation at 95 DEG C for 20 seconds, annealing at 60 DEG C for 15 seconds, and extension at 72 DEG C for 15 seconds were 27 cycles; And final extension at 72 ° C for 1 minute.

PCR products were collected and prepared with SPARK DNA Sample Prep Kit (Enzymatics, USA). Finally, sequencing was performed using a HiSeq 2500 system (Illumina, USA) to obtain gene sequence information for single site or 10 sites.

( off - Target effect analysis)

Using the MIT CRISPR Design Tool (http://crispr.mit.edu/) and E-CRISP software (http://www.e-crisp.org/E-CRISP/), eight potential Off-target cleavage sites were selected.

From the MIT CRISPR Design Tool, 121 potential off-targets were obtained, and four top off-targets were selected based on the scores (OT-1, 2, 3, and 4).

When using E-CRISP software, we set the parameter "Tolerated edit distance to the target sequence" parameter in 3. Seventeen potential off-targets were obtained and four top off-targets were selected based on Efficacy-Score (E-Score) (OT-5, 6, 7, and 8). Selected off-target sites are shown in Table 2. The PCR primers were designed to amplify the off-target region so that the size of the PCR product was 155-199 bp (Table 1, SEQ IDs 145-160). The off-target area was amplified using Kapa HiFi HotStart ReadyMix and performed under the following cycling conditions: initial denaturation at 95 ° C for 3 minutes; Subsequently, denaturation at 95 DEG C for 20 seconds, annealing at 60 DEG C for 15 seconds, and extension at 72 DEG C for 15 seconds were 27 cycles; And final extension at 72 ° C for 1 minute.

Amplicons were collected and prepared using the SPARK ™ DNA Sample Prep Kit (Enzymatics, USA) and finally sequenced using the HiSeq 2500 system (Illumina, USA).

(Data processing and analysis)

Samples were sequenced on an Illumina HiSeq 2500 system. The low end (phred quality score <25) was cut out and the readings with an average phred quality <20 were removed. To identify the recombination efficiency in the E. coli experiment, the readings with the planned formula were counted. In human cell experiments, the readouts were arranged in the reference human genomic sequence (hg19) using Novoalign V2.07.18 (Novocraft Technologies, Malaysia) and inserted within ± 10 bp of the predicted cleavage site (ie, 3 bp upstream of the PAM sequence) Or missing readings were counted. To analyze the substitution efficiency, the readings containing the formula sequences were counted.

< Example  2> sgR Identification of a single-site dielectric correction of E. coli via DNA

To demonstrate that the sgR-DNA prepared in Example 1 can manipulate the genome, sgR-DNA was tested in E. coli without the NHEJ pathway. We targeted the pre-maturation codon (I239 *) of the galK gene to return to the wild-type sequence in the galK _OFF EcNR2 strain. For this, donor DNA of 63 nt or 93 nt or 123 nt containing the sgRNA-DNA expressed under the control of the T7 promoter, 20 nt of the sgRNA spacer complementary to the target dielectric site, and the wild-type sequence (TAA to TAT); And a PAM equation was designed to inhibit re-breaking after calibration (Fig. 6). The designed sgR-DNA was then constructed by PCR assembly (Figure 4).

To operate the sgR-DNA based manipulation system, galK _OFF cells were electroporated with T7 polymerase expression vector (pN249) and Cas9 expression vector (pET-Cas9) and then electroporation to introduce sgR-DNA 6).

On the other hand, the efficiency of other delivery methods using ssRNA (referred to herein as "linear sgRNA") and linear double stranded DNA expressing ssODN was tested (Fig. 7).

The frequency of genetic corrections due to HR increased with donor length (Fig. 8). In addition, when 123 nt donor was used, 11% efficiency (average of three experiments) was observed in sgR-DNA experiments, but when using linear sgRNA with ssODN, low calibration efficiency (4% Average of the turn experiments) was observed (Figure 9). These results demonstrate that the linear construct of the present invention can be used to modify dielectrics in E. coli.

< Example  3> sgR - Identification of multi-site genetic corrections of E. coli through DNA

Next, we analyzed whether the sgR-DNA construct can be applied to improve multi-site dielectric calibration. Screening with a 11 aro gene targeting (aroA, aroB, aroC, aroD , aroE, aroF, aroG, aroK, aroL, aroM, and aroP) distributed all over the dielectric entire leading to the biosynthesis of aromatic amino acids, 123 nt Donor DNA was designed and constructed. Then, the pool of the sgR-DNA library targeting 11 aro genes was transferred to the cells, and the efficiency was examined according to the same procedure as the single operation (Fig. 10). As a control, pools of libraries composed of linear sgRNAs with ssODN were examined to examine the effect of combining sgRNA and donor DNA in sgR-DNA.

Experimental results showed that HR events were observed at all target sites (Fig. 11, sgR-DNA showed an average 2.5% efficiency whereas linear sgRNA and ssODN showed very low correction-efficiency (0.19%). The reduction in efficiency may be explained by similarity to allosteric inhibition, although one gene position is corrected, but the cell can be killed by the remaining sgRNA targeting the other gene (allosteric gene location). The inventors hypothesized that the calibration method using linear sgRNA and ssODN would be much more sensitive because the sgRNA and ssODN were dominant with low efficiency and unpaired sgR-DNA always perform sgRNA and matched donor DNA Because.

< Example  4> sgR Identification of a single-site dielectric correction of human cells via DNA

To establish sgR-DNA for the intended SNV generation in mammalian cells, the base substitution of EGFR corresponding to the amino acid change of wild type Leu858 to Arg858, a driver mutation in lung cancer, was targeted. To this end, we transformed into sgR-DNA regulated by the human transcriptional promoter (Figure 12), three introduced a planned amino acid change (i.e., p.L858R) and the other introduced a PAM formula similar to E. coli manipulation Donor DNA containing four mismatched sequences was designed (Figure 13). Then, sgR-DNA was constructed by PCR assembly (Fig. 5, transferred to HEK293T cells, and the efficiency was examined by NGS. As a control group, efficiency of the transfer method using linear double-stranded DNA expressing sgRNA and ssODN (Fig. 14). In addition, a FACS system was used for cells transfected with Cas9 using GFP.

As a result of NGS, HR-mediated substitution by sgR-DNA showed a yield of 0.6%, demonstrating that sgR-DNA mediates the HR response in mammalian cells (Fig. 15). Unlike the case of Escherichia coli correction, NHEJ-induced indels were predominant and had a high efficiency of 23.9% (FIG. 16). In the Cas9-introduced cells enhanced by FACS, an increased ratio of cells calibrated by sgR-DNA (substitution: 2.0%) was observed compared to the non-selected cells. Similarly, insertion-defect efficiency was also increased.

In addition, genotyping of the target gene position was confirmed in a FACS-isolated monoclone through Sanger sequencing (Figure 17). Of the 43 clones selected, two clones had a mismatch mutation in the heterozygote allele (2.3% of the SNV incidence); Mutant clone # 1 (p.L858R delivers altered alleles and wild-type alleles) and mutant clone # 2 (p.L858R delivers alleles with altered alleles and 12-bp deletions).

< Example  5> off - Target cutting efficiency test

The eight potential off-target sites (OTs) of sgRNAs screened in two off-target prediction tools were identified through NGS.

In the NGS data, basal rate (<0.04%) of insertion-deletion as indicated by the measured sequencing error was observed by plotting the occurrence of insert-defect and the size of insert-defect size at each base position And 20). Insertion-defects are randomly distributed within the periphery of the off-target site, but insertion-defects frequently occur in the expected site of cleavage at the target site (i. E., 3 bp upstream of the PAM sequence). Furthermore, the majority of insert-defects in the off-target region were 1 bp in length, but the size of the insert-defect in the target region was more than tens of bp in length.

In order to test mutations in the efficiency of the sgR-DNA-based method, further, base substitutions of the BRAF gene or the KRAS gene corresponding to the respective amino acid mutations p.V600E and p.G12C were tested.

As a result, substitution efficiencies at the respective gene positions were 0.19% and 0.34% (FIGS. 21 and 22). Based on these results, it was confirmed that any substitution mutations could be introduced by sgR-DNA.

< Example  6> sgR - Verification of multi-site genetic correction of human cells via DNA

It was tested whether the sgR-DNA prepared in Example 4 could be extended to multi-site calibration of mammalian cells. The sgR-DNA-based method was extended to 10 selected gene loci based on high frequencies in the cancer somatic mutation database (COSMIC). Ten selected mutations associated with various cancer types are CTNNB1 p.T41A, DNMT3A p.R882H, GNAQ p.Q209P, GNAS p.R201C, HRAS p.Q61L, IDH2 p.R140Q, NOTCH1 p.R1598P, NRAS p.G12D , PIK3CA p.E545K and TP53 p.R273H (Fig. 23). We constructed individual sgR-DNA and collected to produce the sgR-DNA library. In addition, sgR-DNA-based genome corrections and several control experiments were performed and gene location was analyzed via NGS.

NGS resulted in a total of 0.28% displacement from the sgR-DNA based method and a higher substitution efficiency (0.97%) in the FACS-sorted cell population (Fig. 24). Multiple experimental results suggest that sgR-DNA can be used for multiple location correction, although the efficiency of the method using sgRNA and ssODN is still higher than that of sgR-DNA. SgR-DNA is still expected to be advantageous when the number of gene positions designed to be corrected increases. This is because a decrease in the relative concentration of ssODN will cause a decrease in efficiency when using sgRNA and ssODNs.

In summary, the present invention demonstrates that the sgR-DNA based approach is a new method for the delivery of linear double-stranded DNA encoding sgRNA and donor DNA and is capable of performing multiple HR-mediated genomic modification. The present invention can be used not only in E. coli but also in human genome modification. The system of the present invention is a versatile tool for calibrating both eukaryotes and prokaryotes. In addition, the construction of sgR-DNA is very direct and involves only PCR producing small linear DNA fragments. And although only two oligonucleotides are used as constructs to cover the variable region of 226 nt, which is essential, two oligo for the plasmid and one oligos for the ssODN are required in the preceding method. This eliminates the need for experimental procedures such as sgRNA production and vector cloning for donor DNA production, and reduces costs. It is anticipated that a cleaved oligonucleotide pool can be used from a microarray capable of programming for construction of the sgR-DNA library (Fig. 25).

SEQ ID NO. designation The sequence (5 '- &gt; 3') One galK_spacer GCAGCTTTAACATCTGCCGC 2 aroA_spacer ATTATTTCGAGCAGCTGGCG 3 aroB_spacer CGCTGATTGACAATCGGCAA 4 aroC_spacer TTTTAATGGATCACCTGTTA 5 aroD_spacer GAAATATTATTGCTTATGCC 6 aroE_spacer ACTGGATGGCCTGATTCACG 7 aroF_spacer GGATCTGAACGGGCAGCTGA 8 aroG_spacer CAGTTGACGTAACAGAGCAT 9 aroK_spacer TTTCCAGCATGTGAATAATC 10 aroL_spacer ACAATTGATCGTCTGTGCCA 11 aroM_spacer ATTGATTGCACGGCTGGCTG 12 aroP_spacer ATGCGCTTTTACGGCTTTGG 13 EGFR_spacer GTCTTCCGCACCCAGCAGTT 14 BRAF_spacer GGACAACTGTTCAAACTGAT 15 KRAS_spacer GATGACTGAATATAAACTTG 16 CTNNB1_spacer GAGAGAAGGAGCTGTGGTAG 17 DNMT3A_spacer GGTCTCCAACATGAGCCGCT 18 GNAQ_spacer GGGTCGATGTAGGGGGCCAA 19 GNAS_spacer GCTCAAAGATTCCAGAAGTC 20 HRAS_spacer GCTGGATACCGCCGGCCAGG 21 IDH2_spacer GCCGGAAGACAGTCCCCCCC 22 NOTCH1_spacer GTCACGCTTGAAGACCACT 23 NRAS_spacer GACTGAGTACAAACTGGTGG 24 PIK3CA_spacer GGCTCAGTGATTTCAGAGAG 25 TP53_spacer GCTGGGACGGAACAGCTTTG 26 galK_63_donor_rev CGCGATTTGTGCGCCGTCGAGCGGCAGATGATAAAGCTGCTGCAATACGGTTCCGACCGCGAC 27 galK_93_donor_rev TCCGCTTCACTGGAAGTCGCGGTCGGAACCGTATTGCAGCAGCTTTATCATCTGCCGCTCGACGGCGCACAAATCGCGCTTAACGGTCAGGAA 28 galK_123_donor_rev &Lt; 29 aroA_donor_rev AAAGAAAGATTTGGCTATTTATTGCCCGTTGTTCATTCAGGCTGCCTGGCTAATACGCGCGATCTGCTCGAAATAATCCGGAAATGTTTTGGCCGTGCATTTGGGATCAAGAATCGTCACTGG 30 aroB_donor_rev Gt 31 aroC_donor_rev &Lt; tb & 32 aroD_donor_rev AAAAAAGCGTCTGCGCCAGGGCAAATCTCGGTAAATGATTTGCGCACGGTATTAACTATTATTCATCAGGCATAAGCAATAATATTTCGGCGGGAACACCCTCCCCGCCGAACTAAAAAATAT 33 aroE_donor_rev ATTTTTTATTCTCGTCCCACTCTTCCCTGTCCGGAAACTGGATGGCCTGATTCACGCCGAGATTTCCTCCTGCAATTGCTTTATAACTGGTTCTACGTCAGGCAGAACACCGTGCCAGAGAAG 34 aroF_donor_rev TGATCGCGTAATGCGGTCAATTCAGCAACCATAATAAACCTCTTAAGCCACGCGAGCGGTGATCTGCCCGTTCAGATCCTGATGAATTTCACGCAGCAAGGCATCGGTCATTTCCCAGCTAAT 35 aroG_donor_rev CACTCTGACGGCGCATCCGACAATTAAACCTTACCCGCGACGCGCTTTTACTGCATTCGCGATTTGACGTAACAGAGCATCCGTATCTTCCCAGCCGATGCAGGCATCGGTGATGCTCTTACC 36 aroK_donor_rev ATCCACCTTAATTACTGTACCCGCAGACGAGTGTATATAAAGCCAGAATTAGTTGCTTTCGATCATGTGAATAATCTGATTTGCAACCACTTTAGCGCTTTGATCATCAGTACGAATGGTCAC 37 aroL_donor_rev TATTTCACGGGATGAACGTTAAGTATAGGCGCTCGAAAATCAACAATTGATCGTCTGTGCGATCGCGCTGCGAATTTCAGAAATCACCTGGCTGGGTTCGTTTGTTGCGTCGATGATAATATG 38 aroM_donor_rev TAGTTGAACATCTACTTCACTATAGGGGCCAGAGGCGCTGGCTGTCACGCAAAATTACACGATTAATTCGGCAGCCAGCCGTGCAATCAATACGTTAGACAGCAAGACAGGAACATCGAGCTG 39 aroP_donor_rev Gt 40 EGFR_donor_rev &Lt; RTI ID = 41 BRAF_donor_rev &Lt; 42 KRAS_donor_rev ATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCGCAAGCTCCAACTACCGACAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAAATAA 43 CTNNB1_donor_rev &Lt; 44 DNMT3A_donor_rev GAAGAGGTGGCGGATGACTGGCACGCTCCATGACCGGCCCAGCAGTCTCTGCCTCGCGAAATGGCTCATGTTGGAGACGTCAGTATAGTGGACTGGGAAACCAAATACCCTGGGGGAGAAAAG 45 GNAQ_donor_rev TAGAAACATGATAGAGGTGACATTTTCAAAGCAGTGTATCCATTTTCTTCTCTCTGATCTAGGGCCCCCTACATCGACCATTCTGCAAGGTTAACAATACTCATATTAATAACATATAAAGTA 46 GNAS_donor_rev TTACTGGAAGTTGACTTTGTCCACCTGGAACTTGGTCTCAAAGATTCCAGAAGTCAGCACGCAGCAGCGAAGCAGGTCCTGAAACAAAATTGAGGTCAATGGATCTCACCAAAGCCAACCGAA 47 HRAS_donor_rev CACACACAGGAAGCCCTCCCCGGTGCGCATGTACTGGTCCCGCATGGCGCTGTACTCTTCTAAGCCGGCGGTATCCAGGATGTCCAACAGGCACGTCTCCCCATCAATGACCACCTGCTTCCG 48 IDH2_donor_rev TAGGCGTGGGATGTTTTTGCAGATGATGGGCTCCCGGAAGACAGTCCCCCCCAGAATGTTTTGGATAGTTCCATTGGGACTTTTCCACATCTTCTTCAGCTTGAACTCTGTGAGGACAGAGAT 49 NOTCH1_donor_rev C 50 NRAS_donor_rev ATATTCATCTACAAAGTGGTTCTGGTTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCGTCTGCTCCAACCGACACCAGTTTGTACTCAGTCATTTCACACCAGCAAGAACCTGTTGGAAA 51 PIK3CA_donor_rev GCACTTACTT 52 TP53_donor_rev &Lt; RTI ID = 0.0 &gt; 53 T7_fwd_primer GAAATTAATACGACTCACTATAGG 54 galK_gRNA GAAATTAATACGACTCACTATAGGGCAGCTTTAACATCTGCCGCGTTTTAGAGCTAGAAATAGC 55 gRNA_rev AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC 56 galK_middle CGGAAGAACTTAACCCGGCAAAAAAAGCACCGACTCGG 57 aroA_gRNA GAAATTAATACGACTCACTATAGGATTATTTCGAGCAGCTGGCGGTTTTAGAGCTAGAAATAGC 58 aroB_gRNA GAAATTAATACGACTCACTATAGGCGCTGATTGACAATCGGCAAGTTTTAGAGCTAGAAATAGC 59 aroC_gRNA GAAATTAATACGACTCACTATAGGTTTTAATGGATCACCTGTTAGTTTTAGAGCTAGAAATAGC 60 aroD_gRNA GAAATTAATACGACTCACTATAGGGAAATATTATTGCTTATGCCGTTTTAGAGCTAGAAATAGC 61 aroE_gRNA GAAATTAATACGACTCACTATAGGACTGGATGGCCTGATTCACGGTTTTAGAGCTAGAAATAGC 62 aroF_gRNA GAAATTAATACGACTCACTATAGGGGATCTGAACGGGCAGCTGAGTTTTAGAGCTAGAAATAGC 63 aroG_gRNA GAAATTAATACGACTCACTATAGGCAGTTGACGTAACAGAGCATGTTTTAGAGCTAGAAATAGC 64 aroK_gRNA GAAATTAATACGACTCACTATAGGTTTCCAGCATGTGAATAATCGTTTTAGAGCTAGAAATAGC 65 aroL_gRNA GAAATTAATACGACTCACTATAGGACAATTGATCGTCTGTGCCAGTTTTAGAGCTAGAAATAGC 66 aroM_gRNA GAAATTAATACGACTCACTATAGGATTGATTGCACGGCTGGCTGGTTTTAGAGCTAGAAATAGC 67 aroP_gRNA GAAATTAATACGACTCACTATAGGATGCGCTTTTACGGCTTTGGGTTTTAGAGCTAGAAATAGC 68 aroA_middle GATCAAGAATCGTCACTGGAAAAAAAGCACCGACTCG 69 aroB_middle AATTGCCAACGGAAGAATAAAAAAAGCACCGACTCG 70 aroC_middle CACTGCGCGGATCCCAAAAAAAGCACCGACTCG 71 aroD_middle CCCGCCGAACTAAAAAATATAAAAAAAGCACCGACTCG 72 aroE_middle ACCGTGCCAGAGAAGAAAAAAAGCACCGACTCG 73 aroF_middle CGGTCATTTCCCAGCTAATAAAAAAAGCACCGACTCG 74 aroG_middle TCGGTGATGCTCTTACCAAAAAAAGCACCGACTCG 75 aroK_middle CATCAGTACGAATGGTCACAAAAAAAAGCACCGACTCG 76 aroL_middle GTTGCGTCGATGATAATATGAAAAAAAGCACCGACTCG 77 aroM_middle ACAGGAACATCGAGCTGAAAAAAAAGCACCGACTCG 78 aroP_middle TACGCCCTCACCCGTAAAAAAAGCACCGACTCG 79 U6_fwd_primer AAGGTCGGGCAGGAAGA 80 U6_rev_primer CGGTGTTTCGTCCTTTCCA 81 double-stranded U6 promoter AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG 82 EGFR_gRNA Gt; 83 BRAF_gRNA Gt; 84 KRAS_gRNA Gt; 85 CTNNB1_gRNA Gt; 86 DNMT3A_gRNA Gt; 87 GNAQ_gRNA Gt; 88 GNAS_gRNA Gt; 89 HRAS_gRNA Gt; 90 IDH2_gRNA Gt; 91 NOTCH1_gRNA Gt; 92 NRAS_gRNA Gt; 93 PIK3CA_gRNA Gt; 94 TP53_gRNA Gt; 95 galK_fwd_primer ATCCATGATCCCGCAGTTAC 96 galK_rev_primer GGACATGGTGATCAGCGG 97 aroA_fwd_primer CGCGACATACAATGATCACC 98 aroB_fwd_primer TGCTGCGTGACAAGAAAGTC 99 aroC_fwd_primer GATCACCAAAGGCCGTCAC 100 aroD_fwd_primer AATTTCTCGTCTGGCTGGTG 101 aroE_fwd_primer CAAAGCGTAATGCTGATGGTT 102 aroF_fwd_primer GCGCAGTGAAATGAAATACG 103 aroG_fwd_primer ATCTGGTGGAAGGCAATCAG 104 aroK_fwd_primer ATGAACGCAATCCGCTGTAT 105 aroL_fwd_primer ATGCGCTATATCGCGAAGTT 106 aroM_fwd_primer GTCATCGCGATTTACTGCAA 107 aroP_fwd_primer GGCGGTACTGGTGATTATGC 108 aroA_rev_primer TGACTCACAAGGTCCGAAAA 109 aroB_rev_primer TTCATCCATTTAACACCCCA 110 aroC_rev_primer GCTACTGGCAAGCAGAGCC 111 aroD_rev_primer CCAAATGGAGAATTAGCGCA 112 aroE_rev_primer AGAGATCGCGCATGTCAGTT 113 aroF_rev_primer GTTCCAGACGCTTCGCTAAT 114 aroG_rev_primer TTATCAGGCCTGTGGTGATTC 115 aroK_rev_primer GTTCCCCGAGAGTAACGACA 116 aroL_rev_primer TTACCGTCTGTCCTGGCTTT 117 aroM_rev_primer TGGAAAAGCCGATTGATTTC 118 aroP_rev_primer GTCATCGCGATTTACTGCAA 119 EGFR_fwd_primer GCAGCGGGTTACATCTTCTTTC 120 BRAF_fwd_primer GCCAAAAATTTAATCAGTGGAAAAAA 121 KRAS_fwd_primer CTGCACCAGTAATATGCATATTAAA 122 EGFR_rev_primer CAATACAGCTAGTGGGAAGGCA 123 BRAF_rev_primer ACACATTTCAAGCCCCAAAAA 124 KRAS_rev_primer TAAGCGTCGATGGAGGAGTT 125 CTNNB1_fwd_primer GCTGATTTGATGGAGTTGGAC 126 DNMT3A_fwd_primer CCATGTCCCTTACACACACG 127 GNAQ_fwd_primer CTGACTCCACGAGAACTTG 128 GNAS_fwd_primer CTACTCCAGACCTTTGCTTTAG 129 HRAS_fwd_primer GTCTTTTGAGGACATCCACC 130 IDH2_fwd_primer CGTGCCTGCCAATGGTGA 131 NOTCH1_fwd_primer TTGATGGGGTGCTTGCGC 132 NRAS_fwd_primer TGATCCGACAAGTGAGAGACA 133 PIK3CA_fwd_primer CTGTGAATCCAGAGGGGAAA 134 TP53_fwd_primer TGCTTACCTCGCTTAGTGCTC 135 CTNNB1_rev_primer TGAAGGACTGAGAAAATCCCT 136 DNMT3A_rev_primer GCTGTGTGGTTAGACGGCTT 137 GNAQ_rev_primer CCCTAAGTTTGTAAGTAGTGCT 138 GNAS_rev_primer TCAAGAAACCATGATCTCTGTT 139 HRAS_rev_primer GAAGGTCCTGAGGGGGTC 140 IDH2_rev_primer ATTCTGGTTGAAAGATGGCG 141 NOTCH1_rev_primer GGACTGTGCGGAGCATGTA 142 NRAS_rev_primer AGCTTTAAAGTACTGTAGATGTGGC 143 PIK3CA_rev_primer TGCTGAGATCAGCCAAATTC 144 TP53_rev_primer TTAAATGGGACAGGTAGGACC 145 OT-1_fwd_primer ACTGACAGAAGAAAAACCCGAGAC 146 OT-2_fwd_primer AGCAATGTGGTGGAAGCAAT 147 OT-3_fwd_primer CACCAATTGAAAAACCAGCTTTA 148 OT-4_fwd_primer GAGAATCTCATTTCTTTCAAAATGC 149 OT-5_fwd_primer GAAAATAAGCCATTGTTTTGACC 150 OT-6_fwd_primer GAGGGTTCATTATCATCCTAGTCA 151 OT-7_fwd_primer GGCCACAGAGGTGAGAAGAG 152 OT-8_fwd_primer CCACCCTTGCTTCTACCCTT 153 OT-1_rev_primer GCTCCCTAGCCTGGGTAACT 154 OT-2_rev_primer CAGCCAGGTAGAGGGAAAAAG 155 OT-3_rev_primer GGGAAAAACCAAACACCAAGTA 156 OT-4_rev_primer CAGAGCATTTTGGCAAATCA 157 OT-5_rev_primer TGGCCTCCTGCTCTAGCAGT 158 OT-6_rev_primer CCAAAGTACAAACATGAAGCTG 159 OT-7_rev_primer TGGATATTCACTGTGGCAGG 160 OT-8_rev_primer CCTCAGCAATGAGAGTTCCC

chromosome piece location order The number of mismatches between the EGFR target sequence and the corresponding sequence 19 - 38916086 TTCCTCCCCACCCAGCAGTTTAG 3 20 + 48177634 TTGTTCCACACCCAGCAGTTCAG 3 17 - 40723680 GTCTCCCGGGCCCAGCAGTTCAG 3 5 + 43514894 GTCTTCCACTCACAGCAGTTCGG 3 4 - 72942318 TTCTTCCACACACAGCAGTTTGG 3 16 - 60869491 ATCTTCCTCACCCTGCAGTTAAG 3 One - 48382921 GACTTCAGCACCCAGCATTTCAG 3 17 - 72766545 GGCTTCCCCACCCAGCAGGTGAG 3

<110> University-Industry Foundation, Yonsei University <120> Targeted genome editing based on CRISPR / Cas9 system using short          linearized double-stranded DNA <130> P16U16C0909 <150> 2015-0066054 <151> 2015-05-12 <160> 160 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> galK_spacer <400> 1 gcagctttaa catctgccgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroA_spacer <400> 2 attatttcga gcagctggcg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroB_spacer <400> 3 cgctgattga caatcggcaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroC_spacer <400> 4 ttttaatgga tcacctgtta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroD_spacer <400> 5 gaaatattat tgcttatgcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroE_spacer <400> 6 actggatggc ctgattcacg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroF_spacer <400> 7 ggatctgaac gggcagctga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroG_spacer <400> 8 cagttgacgt aacagagcat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroK_spacer <400> 9 tttccagcat gtgaataatc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroL_spacer <400> 10 acaattgatc gtctgtgcca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroM_spacer <400> 11 attgattgca cggctggctg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroP_spacer <400> 12 atgcgctttt acggctttgg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFR_spacer <400> 13 gtcttccgca cccagcagtt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BRAF_spacer <400> 14 ggacaactgt tcaaactgat 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KRAS_spacer <400> 15 gatgactgaa tataaacttg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CTNNB1_spacer <400> 16 gagagaagga gctgtggtag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNMT3A_spacer <400> 17 ggtctccaac atgagccgct 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GNAQ_spacer <400> 18 gggtcgatgt agggggccaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GNAS_spacer <400> 19 gctcaaagat tccagaagtc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HRAS_spacer <400> 20 gctggatacc gccggccagg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IDH2_spacer <400> 21 gccggaagac agtccccccc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NOTCH1_spacer <400> 22 gtcacgcttg aagaccacgt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRAS_spacer <400> 23 gactgagtac aaactggtgg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PIK3CA_spacer <400> 24 ggctcagtga tttcagagag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TP53_spacer <400> 25 gctgggacgg aacagctttg 20 <210> 26 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> galK_63_donor_rev <400> 26 cgcgatttgt gcgccgtcga gcggcagatg ataaagctgc tgcaatacgg ttccgaccgc 60 gac 63 <210> 27 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> galK_93_donor_rev <400> 27 tccgcttcac tggaagtcgc ggtcggaacc gtattgcagc agctttatca tctgccgctc 60 gacggcgcac aaatcgcgct taacggtcag gaa 93 <210> 28 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> galK_123_donor_rev <400> 28 gccgggttaa gttcttccgc ttcactggaa gtcgcggtcg gaaccgtatt gcagcagctt 60 tatcatctgc cgctcgacgg cgcacaaatc gcgcttaacg gtcaggaagc agaaaaccag 120 ttt 123 <210> 29 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroA_donor_rev <400> 29 aaagaaagat ttggctattt attgcccgtt gttcattcag gctgcctggc taatacgcgc 60 gatctgctcg aaataatccg gaaatgtttt ggccgtgcat ttgggatcaa gaatcgtcac 120 tgg 123 <210> 30 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroB_donor_rev <400> 30 gcttgataag cggcctgacc tttcttgttg ttacgctgat tgacaatcgg caatcgcgtt 60 gataacaagc tcgtgcgaaa cgccgctgcg aacttcactc ttaccaattg ccaacggaag 120 aat 123 <210> 31 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroC_donor_rev <400> 31 attcattttt taccagcgtg gaatatcagt cttcacatcg gcattttgcg cccgttgacg 60 aatcaggtga tccattaaaa cgatcgccag catcgcttct gcgatcggca ctgcgcggat 120 ccc 123 <210> 32 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroD_donor_rev <400> 32 aaaaaagcgt ctgcgccagg gcaaatctcg gtaaatgatt tgcgcacggt attaactatt 60 attcatcagg cataagcaat aatatttcgg cgggaacacc ctccccgccg aactaaaaaa 120 taste 123 <210> 33 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroE_donor_rev <400> 33 attttttatt ctcgtcccac tcttccctgt ccggaaactg gatggcctga ttcacgccga 60 gatttcctcc tgcaattgct ttataactgg ttctacgtca ggcagaacac cgtgccagag 120 aag 123 <210> 34 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroF_donor_rev <400> 34 tgatcgcgta atgcggtcaa ttcagcaacc ataataaacc tcttaagcca cgcgagcggt 60 gatctgcccg ttcagatcct gatgaatttc acgcagcaag gcatcggtca tttcccagct 120 aat 123 <210> 35 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroG_donor_rev <400> 35 cactctgacg gcgcatccga caattaaacc ttacccgcga cgcgctttta ctgcattcgc 60 gatttgacgt aacagagcat ccgtatcttc ccagccgatg caggcatcgg tgatgctctt 120 acc 123 <210> 36 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroK_donor_rev <400> 36 atccacctta attactgtac ccgcagacga gtgtatataa agccagaatt agttgctttc 60 gatcatgtga ataatctgat ttgcaaccac tttagcgctt tgatcatcag tacgaatggt 120 cac 123 <210> 37 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroL_donor_rev <400> 37 tatttcacgg gatgaacgtt aagtataggc gctcgaaaat caacaattga tcgtctgtgc 60 gatcgcgctg cgaatttcag aaatcacctg gctgggttcg tttgttgcgt cgatgataat 120 atg 123 <210> 38 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroM_donor_rev <400> 38 tagttgaaca tctacttcac tataggggcc agaggcgctg gctgtcacgc aaaattacac 60 gattaattcg gcagccagcc gtgcaatcaa tacgttagac agcaagacag gaacatcgag 120 ctg 123 <210> 39 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> aroP_donor_rev <400> 39 ggaatggcga tttcggtata cctgatcccg gtatggctga tcgtgttagg tatcggctat 60 atctttaaag agaaaacggc caaagccgta aaagcgcatt aatctctcta cgccctcacc 120 cgt 123 <210> 40 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> EGFR_donor_rev <400> 40 cctccttact ttgcctcctt ctgcatggta ttctttctct tccgcaccca gcagtttagc 60 tctcccaaaa tctgtgatct tgacatgctg cggtgttttc accagtacgt tcctggctgc 120 cag 123 <210> 41 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> BRAF_donor_rev <400> 41 catccacaaa atggatccag acaactgttc aaactgatga gacccactcc atcgagattt 60 ttctgtagct agaccaaaat cacctatttt tactgtgagg tcttcatgaa gaaatatatc 120 tga 123 <210> 42 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> KRAS_donor_rev <400> 42 atattcgtcc acaaaatgat tctgaattag ctgtatcgtc aaggcactct tgcctacgcc 60 gcaagctcca actaccgaca gtttatattc agtcattttc agcaggcctt ataataaaaa 120 taa 123 <210> 43 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> CTNNB1_donor_rev <400> 43 ttgggaggta tccacatcct cttcctcagg attgccttta ccactcagag aaggagctgt 60 agcagtagca ccagaatgga ttccagagtc caggtaagac tgttgctgcc agtgactaac 120 agc 123 <210> 44 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> DNMT3A_donor_rev <400> 44 gaagaggtgg cggatgactg gcacgctcca tgaccggccc agcagtctct gcctcgcgaa 60 atggctcatg ttggagacgt cagtatagtg gactgggaaa ccaaataccc tgggggagaa 120 aag 123 <210> 45 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> GNAQ_donor_rev <400> 45 tagaaacatg atagaggtga cattttcaaa gcagtgtatc cattttcttc tctctgatct 60 agggccccct acatcgacca ttctgcaagg ttaacaatac tcatattaat aacatataaa 120 gta 123 <210> 46 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> GNAS_donor_rev <400> 46 ttactggaag ttgactttgt ccacctggaa cttggtctca aagattccag aagtcagcac 60 gcagcagcga agcaggtcct gaaacaaaat tgaggtcaat ggatctcacc aaagccaacc 120 gaa 123 <210> 47 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> HRAS_donor_rev <400> 47 cacacacagg aagccctccc cggtgcgcat gtactggtcc cgcatggcgc tgtactcttc 60 taagccggcg gtatccagga tgtccaacag gcacgtctcc ccatcaatga ccacctgctt 120 ccg 123 <210> 48 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> IDH2_donor_rev <400> 48 taggcgtggg atgtttttgc agatgatggg ctcccggaag acagtccccc ccagaatgtt 60 ttggatagtt ccattgggac ttttccacat cttcttcagc ttgaactctg tgaggacaga 120 gat 123 <210> 49 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> NOTCH1_donor_rev <400> 49 ggggaagatc atctgctggc cgtgtgcgtc acgcttgaag accacgtttg tgtgcagcac 60 agggctgagc tcccgcagga agtggaagga gctgttgcgc agctgctccg gcggcatcag 120 cac 123 <210> 50 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> NRAS_donor_rev <400> 50 atattcatct acaaagtggt tctggattag ctggattgtc agtgcgcttt tcccaacacc 60 gtctgctcca accgacacca gtttgtactc agtcatttca caccagcaag aacctgttgg 120 aaa 123 <210> 51 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> PIK3CA_donor_rev <400> 51 gcacttacct gtgactccat agaaaatctt tctcctgctc agtgatttca gagagagaat 60 tttgtgtaga aattgctttg agctgttctt tgtcattttc ccttaattca ttgtctctag 120 cta 123 <210> 52 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> TP53_donor_rev <400> 52 ccctttcttg cggagattct cttcctctgt gcgccggtct ctcccaggac aggcacaaac 60 gtgcaccttc aagctgttcc gtcccagtag attaccacta ctcaggatag gaaaagagaa 120 gca 123 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> T7_fwd_primer <400> 53 gaaattaata cgactcacta tagg 24 <210> 54 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> galK_gRNA <400> 54 gaaattaata cgactcacta tagggcagct ttaacatctg ccgcgtttta gagctagaaa 60 tagc 64 <210> 55 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> gRNA_rev <400> 55 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa 60 cttgctattt ctagctctaa aac 83 <210> 56 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> galK_middle <400> 56 cggaagaact taacccggca aaaaaagcac cgactcgg 38 <210> 57 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroA_gRNA <400> 57 gaaattaata cgactcacta taggattatt tcgagcagct ggcggtttta gagctagaaa 60 tagc 64 <210> 58 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroB_gRNA <400> 58 gaaattaata cgactcacta taggcgctga ttgacaatcg gcaagtttta gagctagaaa 60 tagc 64 <210> 59 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroC_gRNA <400> 59 gaaattaata cgactcacta taggttttaa tggatcacct gttagtttta gagctagaaa 60 tagc 64 <210> 60 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroD_gRNA <400> 60 gaaattaata cgactcacta tagggaaata ttattgctta tgccgtttta gagctagaaa 60 tagc 64 <210> 61 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroE_gRNA <400> 61 gaaattaata cgactcacta taggactgga tggcctgatt cacggtttta gagctagaaa 60 tagc 64 <210> 62 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroF_gRNA <400> 62 gaaattaata cgactcacta taggggatct gaacgggcag ctgagtttta gagctagaaa 60 tagc 64 <210> 63 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroG_gRNA <400> 63 gaaattaata cgactcacta taggcagttg acgtaacaga gcatgtttta gagctagaaa 60 tagc 64 <210> 64 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroK_gRNA <400> 64 gaaattaata cgactcacta taggtttcca gcatgtgaat aatcgtttta gagctagaaa 60 tagc 64 <210> 65 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroL_gRNA <400> 65 gaaattaata cgactcacta taggacaatt gatcgtctgt gccagtttta gagctagaaa 60 tagc 64 <210> 66 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroM_gRNA <400> 66 gaaattaata cgactcacta taggattgat tgcacggctg gctggtttta gagctagaaa 60 tagc 64 <210> 67 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> aroP_gRNA <400> 67 gaaattaata cgactcacta taggatgcgc ttttacggct ttgggtttta gagctagaaa 60 tagc 64 <210> 68 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> aroA_middle <400> 68 gatcaagaat cgtcactgga aaaaaagcac cgactcg 37 <210> 69 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> aroB_middle <400> 69 aattgccaac ggaagaataa aaaaagcacc gactcg 36 <210> 70 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> aroC_middle <400> 70 cactgcgcgg atcccaaaaa aagcaccgac tcg 33 <210> 71 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> aroD_middle <400> 71 cccgccgaac taaaaaatat aaaaaaagca ccgactcg 38 <210> 72 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> aroE_middle <400> 72 accgtgccag agaagaaaaa aagcaccgac tcg 33 <210> 73 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> aroF_middle <400> 73 cggtcatttc ccagctaata aaaaaagcac cgactcg 37 <210> 74 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aroG_middle <400> 74 tcggtgatgc tcttaccaaa aaaagcaccg actcg 35 <210> 75 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> aroK_middle <400> 75 catcagtacg aatggtcaca aaaaaagcac cgactcg 37 <210> 76 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> aroL_middle <400> 76 gttgcgtcga tgataatatg aaaaaaagca ccgactcg 38 <210> 77 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aroM_middle <400> 77 acaggaacat cgagctgaaa aaaagcaccg actcg 35 <210> 78 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> aroP_middle <400> 78 tacgccctca cccgtaaaaa aagcaccgac tcg 33 <210> 79 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> U6_fwd_primer <400> 79 aaggtcgggc aggaaga 17 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> U6_rev_primer <400> 80 cggtgtttcg tcctttcca 19 <210> 81 <211> 265 <212> DNA <213> Artificial Sequence <220> <223> double-stranded U6 promoter <400> 81 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac 60 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa 120 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 180 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 240 atcttgtgga aaggacgaaa caccg 265 <210> 82 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> EGFR_gRNA <400> 82 gtggaaagga cgaaacaccg tcttccgcac ccagcagttg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttctggcagc caggaacgta 140 <210> 83 <211> 148 <212> DNA <213> Artificial Sequence <220> <223> BRAF_gRNA <400> 83 gtggaaagga cgaaacaccg gacaactgtt caaactgatg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 tttcagatat atttcttcat gaagacct 148 <210> 84 <211> 149 <212> DNA <213> Artificial Sequence <220> <223> KRAS_gRNA <400> 84 gtggaaagga cgaaacaccg atgactgaat ataaacttgg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttttattttt attataaggc ctgctgaaa 149 <210> 85 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> CTNNB1_gRNA <400> 85 gtggaaagga cgaaacaccg agagaaggag ctgtggtagg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttgctgttag tcactggcag 140 <210> 86 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> DNMT3A_gRNA <400> 86 gtggaaagga cgaaacaccg gtctccaaca tgagccgctg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttcttttctc ccccagggta 140 <210> 87 <211> 157 <212> DNA <213> Artificial Sequence <220> <223> GNAQ_gRNA <400> 87 gtggaaagga cgaaacaccg ggtcgatgta gggggccaag ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 tttactttat atgttattaa tatgagtatt gttaacc 157 <210> 88 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> GNAS_gRNA <400> 88 gtggaaagga cgaaacaccg ctcaaagatt ccagaagtcg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttttcggttg gctttggtga 140 <210> 89 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> HRAS_gRNA <400> 89 gtggaaagga cgaaacaccg ctggataccg ccggccaggg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttcggaagca ggtggtcatt 140 <210> 90 <211> 142 <212> DNA <213> Artificial Sequence <220> <223> IDH2_gRNA <400> 90 gtggaaagga cgaaacaccg ccggaagaca gtcccccccg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttatctctgt cctcacagag tt 142 <210> 91 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> NOTCH1_gRNA <400> 91 gtggaaagga cgaaacaccg tcacgcttga agaccacgtg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 ttgtgctgat gccgccggag 140 <210> 92 <211> 142 <212> DNA <213> Artificial Sequence <220> <223> NRAS_gRNA <400> 92 gtggaaagga cgaaacaccg actgagtaca aactggtggg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 tttttccaac aggttcttgc tg 142 <210> 93 <211> 146 <212> DNA <213> Artificial Sequence <220> <223> PIK3CA_gRNA <400> 93 gtggaaagga cgaaacaccg gctcagtgat ttcagagagg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 tttagctaga gacaatgaat taaggg 146 <210> 94 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> TP53_gRNA <400> 94 gtggaaagga cgaaacaccg ctgggacgga acagctttgg ttttagagct agaaatagca 60 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 120 tttgcttctc ttttcctatc ctga 144 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> galK_fwd_primer <400> 95 atccatgatc ccgcagttac 20 <210> 96 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> galK_rev_primer <400> 96 ggacatggtg atcagcgg 18 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroA_fwd_primer <400> 97 cgcgacatac aatgatcacc 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroB_fwd_primer <400> 98 tgctgcgtga caagaaagtc 20 <210> 99 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> aroC_fwd_primer <400> 99 gatcaccaaa ggccgtcac 19 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroD_fwd_primer <400> 100 aatttctcgt ctggctggtg 20 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> aroE_fwd_primer <400> 101 caaagcgtaa tgctgatggt t 21 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroF_fwd_primer <400> 102 gcgcagtgaa atgaaatacg 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroG_fwd_primer <400> 103 atctggtgga aggcaatcag 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroK_fwd_primer <400> 104 atgaacgcaa tccgctgtat 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroL_fwd_primer <400> 105 atgcgctata tcgcgaagtt 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroM_fwd_primer <400> 106 gtcatcgcga tttactgcaa 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroP_fwd_primer <400> 107 ggcggtactg gtgattatgc 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroA_rev_primer <400> 108 tgactcacaa ggtccgaaaa 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroB_rev_primer <400> 109 ttcatccatt taacacccca 20 <210> 110 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> aroC_rev_primer <400> 110 gctactggca agcagagcc 19 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroD_rev_primer <400> 111 ccaaatggag aattagcgca 20 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroE_rev_primer <400> 112 agagatcgcg catgtcagtt 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroF_rev_primer <400> 113 gttccagacg cttcgctaat 20 <210> 114 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> aroG_rev_primer <400> 114 ttatcaggcc tgtggtgatt c 21 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroK_rev_primer <400> 115 gttccccgag agtaacgaca 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroL_rev_primer <400> 116 ttaccgtctg tcctggcttt 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroM_rev_primer <400> 117 tggaaaagcc gattgatttc 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> aroP_rev_primer <400> 118 gtcatcgcga tttactgcaa 20 <210> 119 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EGFR_fwd_primer <400> 119 gcagcgggtt acatcttctt tc 22 <210> 120 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> BRAF_fwd_primer <400> 120 gccaaaaatt taatcagtgg aaaaa 25 <210> 121 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> KRAS_fwd_primer <400> 121 ctgcaccagt aatatgcata ttaaa 25 <210> 122 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EGFR_rev_primer <400> 122 caatacagct agtgggaagg ca 22 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BRAF_rev_primer <400> 123 acacatttca agccccaaaa 20 <210> 124 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KRAS_rev_primer <400> 124 taagcgtcga tggaggagtt 20 <210> 125 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CTNNB1_fwd_primer <400> 125 gctgatttga tggagttgga c 21 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNMT3A_fwd_primer <400> 126 ccatgtccct tacacacacg 20 <210> 127 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GNAQ_fwd_primer <400> 127 ctgactccac gagaacttg 19 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GNAS_fwd_primer <400> 128 ctactccaga cctttgcttt ag 22 <210> 129 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HRAS_fwd_primer <400> 129 gtcttttgag gacatccacc 20 <210> 130 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IDH2_fwd_primer <400> 130 cgtgcctgcc aatggtga 18 <210> 131 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NOTCH1_fwd_primer <400> 131 ttgatggggt gcttgcgc 18 <210> 132 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NRAS_fwd_primer <400> 132 tgatccgaca agtgagagac a 21 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PIK3CA_fwd_primer <400> 133 ctgtgaatcc agaggggaaa 20 <210> 134 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TP53_fwd_primer <400> 134 tgcttacctc gcttagtgct c 21 <210> 135 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CTNNB1_rev_primer <400> 135 tgaaggactg agaaaatccc t 21 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNMT3A_rev_primer <400> 136 gctgtgtggt tagacggctt 20 <210> 137 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GNAQ_rev_primer <400> 137 ccctaagttt gtaagtagtg ct 22 <210> 138 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GNAS_rev_primer <400> 138 tcaagaaacc atgatctctg tt 22 <210> 139 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HRAS_rev_primer <400> 139 gaaggtcctg agggggtc 18 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IDH2_rev_primer <400> 140 attctggttg aaagatggcg 20 <210> 141 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NOTCH1_rev_primer <400> 141 ggactgtgcg gagcatgta 19 <210> 142 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NRAS_rev_primer <400> 142 agctttaaag tactgtagat gtggc 25 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PIK3CA_rev_primer <400> 143 tgctgagatc agccaaattc 20 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TP53_rev_primer <400> 144 ttaaatggga caggtaggac c 21 <210> 145 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OT-1_fwd_primer <400> 145 actgacagaa gaaaaacccg agac 24 <210> 146 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-2_fwd_primer <400> 146 agcaatgtgg tggaagcaat 20 <210> 147 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OT-3_fwd_primer <400> 147 caccaattga aaaaccagct tta 23 <210> 148 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> OT-4_fwd_primer <400> 148 gagaatctca tttctttcaa aatgc 25 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OT-5_fwd_primer <400> 149 gaaaataagc cattgttttg acc 23 <210> 150 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OT-6_fwd_primer <400> 150 gagggttcat tatcatccta gtca 24 <210> 151 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-7_fwd_primer <400> 151 ggccacagag gtgagaagag 20 <210> 152 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-8_fwd_primer <400> 152 ccacccttgc ttctaccctt 20 <210> 153 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-1_rev_primer <400> 153 gctccctagc ctgggtaact 20 <210> 154 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OT-2_rev_primer <400> 154 cagccaggta gagggaaaaa g 21 <210> 155 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OT-3_rev_primer <400> 155 gggaaaaacc aaacaccaag ta 22 <210> 156 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-4_rev_primer <400> 156 cagagcattt tggcaaatca 20 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-5_rev_primer <400> 157 tggcctcctg ctctagcagt 20 <210> 158 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OT-6_rev_primer <400> 158 ccaaagtaca aacatgaagc tg 22 <210> 159 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-7_rev_primer <400> 159 tggatattca ctgtggcagg 20 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OT-8_rev_primer <400> 160 cctcagcaat gagagttccc 20

Claims (14)

A promoter for expressing the guide RNA;
DNA encoding guide RNA;
Terminator; And
Linear double stranded DNA containing donor DNA.
The method according to claim 1,
Linear double stranded DNA comprises a promoter for expressing the guide RNA; DNA encoding guide RNA; Terminator; And donor DNA are sequentially linked.
The method according to claim 1,
Wherein the promoter comprises any one of T7 promoter, SP6 promoter, rpr-1 promoter, rrk promoter or U6 promoter.
The method according to claim 1,
The guide RNA comprises a spacer recognizing the target DNA; And a guide RNA scaffold.
5. The method of claim 4,
The target DNA is an intrinsic target DNA, linear double stranded DNA.
5. The method of claim 4,
The spacer comprises a portion or all of the sequence of the protospacer adjacent motifs (PAMs).
5. The method of claim 4,
The guide RNA scaffold is a single-chain guide RNA, linear double stranded DNA.
8. The method of claim 7,
Single-stranded guide RNA contains a portion of the crRNA and tracrRNA, linear double-stranded DNA.
The method according to claim 1,
The terminator is either the RNA Polymerase III terminator or the -TTTTTT-sequence, linear double stranded DNA.
The method according to claim 1,
Wherein the donor DNA comprises a PAM sequence and consists of a homologous sequence comprising a mutation codon having a mismatch of 1 to 3 nucleotides with respect to the target DNA.
A linear double stranded DNA of claim 1; And
A composition for CRISPR-Cas9-based genetic correction comprising a Cas9 protein or a vector expressing Cas9 protein.
12. The method of claim 11,
Linear double-stranded DNA and a vector expressing Cas9 protein or Cas9 protein are delivered via co-transfection or serial-transfection to prokaryotic or eukaryotic cells, a CRISPR-Cas9 based genetic correction / RTI &gt;
13. The method of claim 12,
Wherein stepwise transfection comprises transfecting a prokaryotic or eukaryotic cell with a vector expressing Cas9 protein or Cas9 protein, followed by transfection of linear double stranded DNA.
12. The method of claim 11,
Wherein the composition induces a targeted mutation of a single site or multiple location gene in prokaryotic or eukaryotic cells.

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