METHOD FOR SINGLE-BASE GENOME EDITING USING CRISPR/CPF1 SYSTEM AND USES THEREOF

Abstract

The present disclosure relates to a method of editing a genome based on the CRISPR/Cpf1 system and a use thereof, and the CRISPR system using an oligonucleotide-induced mutation and 3′-truncated crRNA according to the present disclosure provides the significant effect of genome editing to the target DNA and thus it is expected that the CRISPR system of the present disclosure may be used in a wide range of fields such as a composition for gene editing using gene scissors, screening at the genome level, therapeutics for various diseases including cancer, development of a composition for disease diagnosis or imaging, and development of transgenic animals and plants.

Claims

1. A method for single-base genome editing based on a CRISPR/Cpf1 system comprising crRNA (CRISPR RNA) and a donor nucleic acid molecule that complementarily binds to a target DNA, the method comprising preparing a 3′-truncated crRNA in which 1 to 5 nucleotides are truncated from the 3′-end of the crRNA comprising a nucleotide sequence complementary to the target DNA.

2. The method of claim 1, wherein the 3′-end-truncated crRNA comprises a region consisting of 15 to 20 consecutive nucleotides complementary to the target DNA.

3. The method of claim 1, wherein the target DNA comprises a nucleotide of a sequence complementary to the crRNA and a protospacer-adjacent motif (PAM).

4. The method of claim 1, wherein the donor nucleic acid molecule is in single-stranded or double-stranded form.

5. The method of claim 1, wherein the donor nucleic acid molecule induces a genetic modification on the target DNA.

6. The method of claim 5, wherein the modifications include a substitution of one or more nucleotides, an insertion of one or more nucleotides, a deletion of one or more nucleotides, a knockout, a knockin, a replacement of an endogenous nucleic acid sequence with a homologous, orthologous, or heterologous nucleic acid sequence, or a combination thereof.

7. A method for increasing genome editing efficiency based on a CRISPR/Cpf1 system comprising crRNA (CRISPR RNA) and a donor nucleic acid molecule that complementarily binds to a target DNA, the method comprising preparing a 3′-truncated crRNA in which 1 to 5 nucleotides are truncated from the 3′-end of the crRNA comprising a nucleotide sequence complementary to the target DNA.

8. A method for preparing a subject in which a target DNA is edited based on the CRISPR/Cpf1 system, comprising the steps of: (a) constructing a donor nucleic acid molecule that complementarily binds to the target DNA and induces modification on the target DNA; (b) constructing a 3′-truncated crRNA in which 1 to 5 nucleotides are truncated from the 3′-end of the crRNA comprising a nucleotide sequence complementary to the target DNA; and (c) contacting the donor nucleic acid molecule of step (a) and the 3′-truncated crRNA of step (b) into the subject to be edited, thereby editing the target DNA of the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1A shows a conceptual diagram of constructing a negative selection system using CRISPR/Cpf1, FIG. 1B shows the low efficiency of single and double base editing using oligonucleotide-induced mutation, and FIG. 1C shows the mismatch-tolerant properties of CRISPR/Cpf1.

[0066] FIG. 2 shows a Sanger sequence analysis result of a target site after base editing, indicating the inaccuracy of single base editing.

[0067] FIGS. 3A and 3B are graphs showing cleavage tolerance in galK(A) and xylB(B) genes using 3′-end truncated crRNA and the ability to discriminate single base mismatches at the maximum length of 3′-end cleavage, respectively, and FIG. 3C shows a conceptual diagram of the same.

[0068] FIG. 4A shows a conceptual diagram applied to single base editing based on FIG. 3, and FIGS. 4B and 4C are graphs showing the improvement of single base editing ability of the actual 3′-end truncated crRNA.

[0069] FIG. 5 shows a Sanger sequence analysis result of the single base editing target site of FIG. 4, indicating the accuracy of single base editing of the 3′-end truncated crRNA.

[0070] FIGS. 6A and 6B are tables showing actual base editing results through sequence analysis of randomly selected galK(A) and xylB(B) targets, respectively, after single base editing, and FIG. 6C is a graph showing the success rate according to the type of mutation, respectively.

[0071] FIG. 7 shows the nucleotide sequence analysis results of randomly selected galK targets after single base editing performed in FIG. 6A.

[0072] FIG. 8 shows the nucleotide sequence analysis results of xylB targets randomly selected after single base editing performed in FIG. 6B.

[0073] FIGS. 9A and 9B are graphs showing the results of single nucleotide insertion/deletion editing of galK(A) and xylB(B) genes using 3′-end truncated crRNA.

[0074] FIG. 10 shows a nucleotide sequence analysis result of a target site after single nucleotide insertion/deletion editing in FIG. 9.

DETAILED DESCRIPTION

[0075] In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0076] Hereinafter, the examples are only for explaining the present disclosure in more detail, and it will be apparent to those of ordinary skill in the art to which the present disclosure belongs that the scope of the present disclosure is not limited by these embodiments according to the gist of the present disclosure.

Example 1. Construction of a Negative Selection System using CRISPR/Cpf1

[0077] The present inventors produced a mutant E. coli strain (E. coli MG1655 araBAD::P.sub.BAD-cpf1-KmR) in which the Cpf1 gene is inserted into the genome through lambda-red recombineering. The E. coli MG1655 strain was spread on LB solid medium and a single colony grown was inoculated in 200 ml of LB liquid medium. They were cultured at 37° C. until OD.sub.600 nm approached 0.4 and centrifuged at 3500 rpm for 20 minutes. They were washed with 40 ml of 10% glycerol twice to prepare electrocompetent cells.

[0078] The cpf1 gene to be inserted was PCR-amplified from pJYS1Ptac (Addgene plasmid #85545) and was amplified by PCR to be the cpf1-KmR cassette to have a homologous sequence for recombination together with the kanamycin gene. The amplified PCR product was purified and then inserted into E. coli MG1655 overexpressed with the lambda-red recombinase of the pKD46 plasmid by L-arabinose to be located at the back of the promoter in which the expression of the gene in L-arabinose is induced, thereby preparing the HK1061 strains.

[0079] After spreading the HK1061 strain on the LB solid medium, the grown single colonies were inoculated into 200 ml of the LB liquid medium and cultured at 30° C. until the OD.sub.600 nm became 0.4. L-arabinose was added at a concentration of 1 mM and further cultured for 3 hours to overexpress the lambda-red beta protein and Cpf1 protein. After centrifugation at 3500 rpm for 20 minutes, washing with 40 ml of 10% glycerol was performed twice to prepare electrocompetent cells.

[0080] Thereafter, the lambda-red beta expression plasmid pHK463 to aid recombination by oligonucleotide was inserted into the HK1061 strain of Example 1. The single colony formed after plating was inoculated into 200 ml of LB solid medium and cultured until OD.sub.600 nm became 0.4 at 30° C. L-arabinose was added at a concentration of 1 mM and further cultured for 3 hours to overexpress the lambda-red beta protein and Cpf1 protein. After centrifugation at 3500 rpm for 20 minutes, washing with 40 ml of 10% glycerol was performed twice to prepare electrocompetent cells. After electroporation, galactose was plated on a MacConkey plate selective medium with 5 g/L of galactose and cultured at 37° C.

[0081] When the crRNA plasmid is inserted into the HK1061 strain overexpressing the Cpf1 protein, the crRNA/Cpf1 complex is formed, and a double-stranded break occurs in the target DNA sequence complementary to the crRNA. When a double-stranded break occurs, E. coli is killed because it does not have a system to repair the break. The reduction in CFU resulting from cell death may determine whether the CRISPR/Cpf1 gene scissors work.

[0082] When a mutation is introduced into the target DNA sequence of the gene scissors by oligonucleotide, the sequence into which the mutation is introduced is not recognized as the target DNA by the gene scissors so that cells may survive. On the other hand, non-mutagenic target DNA is recognized by gene scissors, and the double-stranded DNA is cleaved, resulting in cell death. This is called negative selection. Through negative selection, the target genome may be effectively edited and selected at the level of a single base.

[0083] The present inventors produced a stop codon at bases 503 to 505 in the galK gene of E. coli (NCBI accession no. 945358) to induce immature synthesis termination of GalK protein. In the case of edited cells, white colonies were formed in McConkey's selective medium containing galactose, and unmutated cells formed red colonies. A system was constructed to estimate the editing efficiency by the ratio of each color of the colonies formed in McConkey's solid medium [white colony/(white colony+red colony)] (FIG. 1A), and the effect of editing 1 to 3 bases by negative selection was confirmed.

[0084] When a plasmid that does not express crRNA was inserted by electroporation, negative selection did not occur, resulting in a CFU level of 10.sup.7/μg DNA (FIG. 1B). As a result of inserting only a plasmid expressing crRNA without an oligonucleotide, it was shown that the CFU was reduced to the level of 10.sup.3/μg DNA by negative selection. It is considered that the crRNA/complex normally causes double-strand breaks in the target DNA, resulting in cell death. When the oligonucleotide and crRNA expression plasmid were inserted together, the single-base or double-base editing efficiency was low at 5% and 7% or less, respectively, whereas the editing efficiency of three bases was 67%, which was significantly higher than that of single and double base editing.

[0085] To check the accuracy of base editing, ten, five, and five colonies formed in white color in McConkey's selective medium were selected and sequenced for each number of base edits. As a result, as shown in FIG. 2, only one of the ten white colonies generated when a single base editing oligonucleotide was inserted correctly changed only the 504 target base, and unwanted additional mutations were observed in the remaining nine colonies. On the other hand, it was confirmed that only the targeted bases were accurately changed in all five colonies in the case of double and triple base editing. When the single base editing efficiency and sequence analysis results were combined, it was confirmed that only 0.5%, which is 1/10 of the 5% formed in McConkey's selective medium, was edited correctly, significantly reducing editing efficiency and accuracy.

[0086] Additionally, in order to confirm the mismatch tolerance of CRISPR/Cpf1, negative selection was performed with a mismatch plasmid in which 1 to 4 mismatched bases and a mismatch were assigned to the crRNA complementary to the target DNA. As a result, it was shown that when there are two or less mismatched bases in the crRNA, the CFU was 10.sup.3/μg DNA level due to negative selection, but when there are 3 or more mismatches in the crRNA, the target was not recognized, and negative selection was not performed so that CFU was significantly increased to 10.sup.6/μDNA or more (FIG. 1C). The results demonstrate the properties of Cpf1 to cause mismatch tolerance between the target DNA and the complementary crRNA, and the difficulty of editing single or double bases.

Example 2. 3′-end Truncation of CRISPR/Cpf1 crRNA and Single Base Mismatch Intolerance

[0087] Previous studies reported that the crRNA/Cpf1 complex may cause double-strand breaks in the target DNA even when the 3′ end of the CRISPR/Cpf1 crRNA is removed by 4 to 6 nucleotides (nt). The present inventors introduced a crRNA plasmid in which the 3′-end of the crRNA was removed by 1 to 6 nt into HK1061 cells to confirm the operation of the gene scissors so that they were intended to confirm the characteristics of the mismatch tolerance and truncation tolerance of CRISPR/Cpf1.

[0088] As a result, as shown in FIGS. 3A to 3B, it was confirmed that the CFU decreased to the level of 10.sup.3/μDNA even when the 3′-end of crRNA was cut by 5 nt. However, when the 3′-end truncation was present for 6 nt or more in crRNA, the CFU was elevated to the level of 10.sup.7/μDNA, probably because the 3′-end 6 nt-truncated crRNA/Cpf1 complex gene scissors did not work. Additionally, 1 to 6 3′-end truncation and single base mismatches were simultaneously given to the crRNA, and it was observed that the 3′-end 6 nt-truncated crRNA did not have mismatch tolerance and could be distinguished from a single mismatch with the target. In the case of crRNA having a single mismatch up to the truncation of 4 nt or less at the 3′ end at the same time, CFU was reduced by negative selection. On the other hand, when a single mismatch and a 3′ 5-nt truncation were simultaneously present, the CFU was significantly elevated at 10.sup.6-7/μg DNA level compared to that only the presence of 3′ 5-nt truncation in crRNA could cause double-stranded break in cells.

[0089] Accordingly, the present inventors applied 3′-end truncated crRNA to a single base editing method based on the result that it was not recognized as a target when both 3′ 5 nt-truncation and a single mismatch exist in crRNA in CRISPR/Cpf1 (FIG. 3C).

TABLE-US-00001 TABLE 1 SEQ Primer sequence ID NO Primer Name (5′.fwdarw.3′) 1 P1 CAATAACTAAGTCCCTTTGA GTGAGCTGATACCGCTCGCC G 2 P2 CAAGAACCAGGACCGGTAAT ACGGTTATCCACAGAATCAG G 3 P3 AACCGTATTACCGGTCCTGG TTCTTGTCCTGGGCAACGTT G 4 P4 GATTCCGCGAACCCCAGAGT CCCGCAGGAGCCTCAAAAAT CGAGCTCG CTTTGGTC 5 P5 CAAAGCGAGCTCGATTTTTG AGGCTCCTGCGGGACTCTGG GGTTCGCG GAATCATG 6 P6 GCTCACTCAAAGGGACTTAG TTATTGCGGTTCTGGACAAA T 7 galK504A GAAAACCAGTTTGTAGGCTG AAACTGCGGGATCATGGATC A 8 galK504_del GAAAACCAGTTTGTAGGCTG AACTGCGGGATCATGGATCA 9 galK504_insC GAAAACCAGTTTGTAGGCTG CTAACTGCGGGATCATGGAT CA 10 galK510_del CAGTTTGTAGGCTGTAACTG GGGATCATGGATCAGCTAAT 11 galK510_insG CAGTTTGTAGGCTGTAACTG GCGGGATCATGGATCAGCTA AT 12 galK505C_F TAGGCTGTCACTGCGGGATC AATTTAAATAAAACGAAAGG CTCAGTC 13 galK505C_R TGATCCCGCAGTGACAGCCT AATCTACAACAGTAGAAATT CGGATCC 14 galK505GG_F TAGGCTGTGGCTGCGGGATC AATTTAAATAAAACGAAAGG CTCAGTC 15 galK505GG_R TGATCCCGCAGCCACAGCCT AATCTACAACAGTAGAAATT CGGATCC 16 galK505CCA_F TAGGCTGTCCATGCGGGATC AATTTAAATAAAACGAAAGG CTCAGTC 17 galK505CCA_R TGATCCCGCATGGACAGCCT AATCTACAACAGTAGAAATT CGGATCC 18 galK_15_F GTAGATTAGGCTGTAACTGC GATTTAAATAAAACGAAAGG CTCAGTC 19 galK_15_R CGCAGTTACAGCCTAATCTA CAACAGTAGAAATTCGGATC C 20 galK_16_F TAGATTAGGCTGTAACTGCG GATTTAAATAAAACGAAAGG CTCAGTC 21 galK_16_R CCGCAGTTACAGCCTAATCT ACAACAGTAGAAATTCGGAT CC 22 galK_17_F AGATTAGGCTGTAACTGCGG GATTTAAATAAAACGAAAGG CTCAGTC 23 galK_17_R CCCGCAGTTACAGCCTAATC TACAACAGTAGAAATTCGGA TCC 24 galK_18_F GATTAGGCTGTAACTGCGGG AATTTAAATAAAACGAAAGG CTCAGTC 25 galK_18_R TCCCGCAGTTACAGCCTAAT CTACAACAGTAGAAATTCGG ATCC 26 galK_19_F ATTAGGCTGTAACTGCGGGA TATTTAAATAAAACGAAAGG CTCAGTC 27 galK_19_R ATCCCGCAGTTACAGCCTAA TCTACAACAGTAGAAATTCG GATCC 28 galK_20_F TTAGGCTGTAACTGCGGGAT CATTTAAATAAAACGAAAGG CTCAGTC 29 galK_20_R GATCCCGCAGTTACAGCCTA ATCTACAACAGTAGAAATTC GGATCC 30 galK505CCAG_ TAGGCTGTCCAGGCGGGATC 21_F AATTTAAATAAAACGAAAGG CTCAGTC 31 galK505CCAG_ TGATCCCGCCTGGACAGCCT 21_R AATCTACAACAGTAGAAATT CGGATCC 32 galK505C_20_F TTAGGCTGTCACTGCGGGAT CATTTAAATAAAACGAAAGG CTCAGTC 33 galK505C_20_R GATCCCGCAGTGACAGCCTA ATCTACAACAGTAGAAATTC GGATCC 34 galK505C_19_F ATTAGGCTGTCACTGCGGGA TATTTAAATAAAACGAAAGG CTCAGTC 35 galK505C_19_R ATCCCGCAGTGACAGCCTAA TCTACAACAGTAGAAATTCG GATCC 36 galK505C_18_F GATTAGGCTGTCACTGCGGG AATTTAAATAAAACGAAAGG CTCAGTC 37 galK505C_18_R TCCCGCAGTGACAGCCTAAT CTACAACAGTAGAAATTCGG ATCC 38 galK505C_17_F AGATTAGGCTGTCACTGCGG GATTTAAATAAAACGAAAGG CTCAGTC 39 galK505C_17_R CCCGCAGTGACAGCCTAATC TACAACAGTAGAAATTCGGA TCC 40 galK505C_16_F TAGATTAGGCTGTCACTGCG GATTTAAATAAAACGAAAGG CTCAGTC 41 galK505C_16_R CCGCAGTGACAGCCTAATCT ACAACAGTAGAAATTCGGAT CC

Example 3. Single Base Editing using 3′-end Truncated crRNA

[0090] Based on the results of Example 2, the present inventors attempted to increase single base editing efficiency by applying to base editing that a single base mismatch is distinguished when the 3′-end truncation is maximally present (FIG. 4A). Mutagenic oligonucleotides were prepared so that one base each of 504 of the galK gene and 643 of the xylB (NCBI accession no. 948133) gene were substituted. The single base editing efficiency of CRISPR/Cpf1 using a 3′-end truncated crRNA was calculated with a ratio by a color of colonies formed in McConkey solid medium after a crRNA expression plasmid and a mutagenic oligonucleotide were electroporated into HK1061 in the same manner as in Example 1.

[0091] In single base editing of the galK gene, when there was a truncation of 4 nt or less at the 3′-end of the crRNA, less than 10% of white colonies were formed due to the mismatch/truncation tolerance of the CRISPR/Cpf1 system (FIG. 4B). In the xylB gene, as the number of 3′-end truncation of crRNA increased from 0 to 4 nt, the percentage of white colonies gradually increased from 4% to 76% (FIG. 4C). In both genes, when a 5 nt truncation was present at the 3′-end of crRNA, both galK 504 base and xylB 643 base showed a significant increase in the proportion of white colonies generated by single base editing to 87%. Thereafter, the sequence analysis confirmed that only base 504 of the galK gene and base 643 of the xylB gene were correctly changed (FIG. 5).

[0092] These results show that the presence of a 5 nt truncation at the 3′-end of the crRNA of CRISPR/Cpf1 induces a double-strand break in the unmutated target, but a target having a single base mismatch due to mutation is not recognized as a target to obtain a single base edited strain.

Example 4. Verification of Single Base Editing Efficiency of 3′-end Truncated crRNA Through Random Candidate Sequencing

[0093] The present inventors tried to confirm whether the ability of the 3′-end truncated crRNA to improve the single base editing efficiency may be applied to various targets other than 504 of galK, and 643 of xylB. In order to perform all possible edits at the base at various positions within the same target DNA sequence N.sub.21, an oligonucleotide was constructed to substitute three different bases except for itself (A.fwdarw.G/T/C, T.fwdarw.G/A/C, G.fwdarw.A/T/C, or C.fwdarw.G/A/T). A total of 8 target bases for each gene were set as two each for G, A, T, and C. Three single base editing oligonucleotides were constructed per position of one target base. Thus, a total of 24 electroporations (=possible base editing) were performed (FIGS. 6A to 6B). Four colonies were randomly selected from the colonies formed after plating on LB medium supplemented with spectinomycin at 75 μgml.sup.−1. Sanger sequencing confirmed the single base editing ability in which when a single base was correctly changed in even one colony, it was considered as a success (FIGS. 7 to 8).

[0094] In order to compare the improvement in single base editing efficiency of 3′-end truncated crRNA, the base editing ability was first confirmed with a crRNA plasmid without 3′-end truncation. The results indicate that only one of 24 base edits was successful in both galK and xylB genes (FIGS. 6A to 6B). On the other hand, when the 3′-end 5 nt truncated crRNA showing the maximum editing efficiency in each gene was used, 79.1% of the galK gene (19 of 24 edited) and 50% of the xylB gene (12 of 24) were shown so that the single base editing ability was significantly improved in both genes. The results of 19 edits of galK and 12 edits of xylB (/24 edits), which were successful in introducing mutations, were analyzed by mutation type (8 transition+16 transversion). In galK, the transition was 62.5% (=⅝) and transversion was 87.5% (= 14/16), respectively. In xylB, transition was 25% (= 2/8) and transversion was 62.5% (= 10/16), respectively. These results indicate that transversion was more predominant in both genes (FIG. 6C). This demonstrates that the 3′-end truncated crRNA of the present disclosure is an optimal condition in which single base editing ability is greatly improved at the maximum number of truncations, and it shows that transversion-type base editing may be performed better.

Example 5. Confirmation of Single Nucleotide Insertion/Deletion Editing Efficiency of 3′-end Truncated crRNA

[0095] The present inventors confirmed whether the 3′-end truncated crRNA affects not only single base editing but also the improvement of single nucleotide insertion or deletion efficiency.

[0096] The brief is as follows.

[0097] When base 509 of the galK gene is deleted, or a single nucleotide is inserted at position 510, a frame shift of the galK gene occurs to generate a stop codon at base 600's, leading to premature translation termination so that the GalK protein is not synthesized normally. Strains with single nucleotide deletion or insertion may not normally metabolize galactose and form white colonies on the McConkey medium. Therefore, it is possible to estimate the efficiency of deletion or insertion of a single nucleotide by checking change in the color of colonies formed in McConkey's medium. In the same principle, a mutation-inducing oligonucleotide was prepared so that base 643 in xylB was deleted or inserted. It was inserted into HK1061 together with crRNA plasmids having 0, 4, 5, and 6 nt truncation at 3′ of crRNA, and the color change of colonies formed in McConkey's medium was observed.

[0098] As a result, in the case of using an untruncated crRNA plasmid, single nucleotide insertion/deletion editing efficiency showed less than 10% in both galK and xylB genes. In the case of the 3′-end 4 nt truncated crRNA plasmid, the single nucleotide insertion efficiency at base 510 of galK was 22%, and the single nucleotide deletion efficiency at base 509 of galK was 19% (FIG. 9A). The insertion efficiency at base 643 of the xylB gene was slightly increased to 20%, and the single nucleotide deletion efficiency at base 643 was slightly increased to 12%.

[0099] Meanwhile, in the case of the 3′-end 5 nt truncated crRNA plasmid, the single nucleotide insertion efficiency at base 510 of galK was significantly increased to 79%, and the nucleotide deletion efficiency at base 509 was significantly increased to 76%. The insertion efficiency of the xylB gene at base 643 was significantly increased to 62%, and the single nucleotide deletion efficiency at base 643 was significantly increased to 58%. The nucleotide sequence analysis confirmed that only the target base was accurately changed (FIG. 10). When the 3′-end 6 nt truncated crRNA plasmid was used, the CFU was elevated to the level of 10.sup.7/μg DNA, regardless of the nucleotide deletion or insertion site. These results show the same trend as in Example 1. These results show that it is most effective for all types of genome editing, including single base editing, insertion, or deletion within the maximum length where the 3′-end truncation of Cpf1 crRNA is allowed.

[0100] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.