JP-7855628-B2 - A composition for cleaving target DNA, comprising a guide RNA specific to the target DNA and a CAS protein-coding nucleic acid or CAS protein, and its use.

Inventors

• キム，ジン－ソー
• チョ，スン ウー
• キム，ソジュン
• キム，ジョン ミン
• キム，ソクジョーン

Assignees

• ツールゲン インコーポレイテッド

Dates

Publication Date

20260508

Application Date

20240422

Priority Date

20121023

Claims (16)

1. A method for inducing modification of a target nucleic acid sequence in higher plant cells, To obtain an artificial and/or non-naturally occurring type II Cas9/RNA complex by preparing a composition comprising recombinant Cas9 protein, guide RNA, and a Cas9/RNA complex formed by at least a portion of the recombinant Cas9 protein and the guide RNA; and to introduce the Cas9/RNA complex into higher plant cells. Includes, The guide RNA, which includes the crRNA and tracrRNA portions, is either in vitro transcribed RNA or synthetic RNA. The target nucleic acid sequence, which is endogenous DNA, includes a portion complementary to the crRNA portion of the guide RNA. In the above composition, the guide RNA is in at least a 2-fold molar excess relative to the recombinant Cas9 protein. The Cas9/RNA complex is a combination of the recombinant Cas9 protein and the guide RNA, and the Cas9/RNA complex forms before being introduced into higher plant cells. The aforementioned method.
2. Guide RNA, (i) Dual guide RNA containing crRNA and tracrRNA; or (ii) Single-stranded guide RNA containing crRNA fused with tracrRNA The method according to claim 1.
3. A composition for use in a method of introducing a Cas9/RNA complex into higher plant cells to induce modification of a target nucleic acid sequence in the higher plant cells, comprising a recombinant Cas9 protein, a guide RNA, and an artificial and/or non-spontaneously occurring type II Cas9/RNA complex formed by at least a portion of the recombinant Cas9 protein and the guide RNA, wherein the complex is (a) Cas9 protein, and (b) Guide RNA containing the crRNA and tracrRNA portions It contains and forms a complex before being introduced into higher plant cells. The aforementioned guide RNA is RNA transcribed in vitro or synthetic RNA. The target nucleic acid sequence, which is endogenous DNA, includes a portion complementary to the crRNA portion of the guide RNA. The guide RNA is in at least a 2-fold molar excess relative to the recombinant Cas9 protein. The aforementioned composition.
4. Guide RNA, (i) Dual guide RNA containing crRNA and tracrRNA; or (ii) Single-stranded guide RNA containing crRNA fused with tracrRNA The composition according to claim 3.
5. The method according to any one of claims 1 to 2, wherein the target nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by Cas9, and the PAM is composed of a trinucleotide 5'-NGG-3'.
6. The composition according to any one of claims 3 to 4, wherein the target nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by Cas9, and the PAM is composed of a trinucleotide 5'-NGG-3'.
7. The method according to any one of claims 1 to 2, wherein the crRNA contains two additional guanine nucleotides at its 5' end.
8. The composition according to any one of claims 3 to 4, wherein the crRNA contains two additional guanine nucleotides at its 5' end.
9. The method according to any one of claims 1 to 2, wherein the Cas9 protein contains a nuclear localization signal (NLS), and the NLS is located at the N-terminus or C-terminus of the Cas9 protein.
10. The composition according to any one of claims 3 to 4, wherein the Cas9 protein contains a nuclear localization signal (NLS), and the NLS is located at the N-terminus or C-terminus of the Cas9 protein.
11. The method according to any one of claims 1 to 2, wherein the crRNA is 20 nucleotides long.
12. The composition according to any one of claims 3 to 4, wherein the crRNA is 20 nucleotides long.
13. The method according to any one of claims 1 to 2, wherein the modification comprises one of at least one nucleotide deletion, insertion, substitution, or indel.
14. The composition according to any one of claims 3 to 4, wherein the modification comprises one of at least one nucleotide deletion, insertion, substitution, or indel.
15. The method according to any one of claims 1 to 2, wherein the molar ratio of guide RNA to recombinant Cas9 protein is 29:14.0 to 29:1.4.
16. The composition according to any one of claims 3 to 4, wherein the molar ratio of guide RNA to recombinant Cas9 protein is 29:14.0 to 29:1.4.

Description

This invention relates to targeted genome editing in eukaryotic cells or eukaryotes. More specifically, the invention relates to a composition for cleaving target DNA in eukaryotic cells or eukaryotes, comprising a guide RNA specific to target DNA and a Cas protein-coding nucleic acid or Cas protein, and to the use thereof. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a locus containing numerous short serial repeats found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as the immune system in prokaryotes, where it confers resistance to exogenous genetic factors such as plasmids and phages. The CRISPR system results in a type of adaptive immunity. Short segments of foreign DNA, called spacers, are incorporated between CRISPR repeats in the genome, functioning as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic factors in a manner similar to RNAi in eukaryotes. Cas9, an essential protein component of the type II CRISPR/Cas system, forms an active endonuclease when it complexes with two RNAs called CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). This endonuclease cleaves invading phages or plasmids, thereby defending host cells. crRNA is transcribed from CRISPR elements in the host genome that have previously been captured from such invaders. Recently, Jinek et al. (1) demonstrated that single-stranded chimeric RNA produced by the fusion of essential regions of crRNA and tracrRNA can replace the two RNAs in the Cas9/RNA complex, forming a functional endonuclease. The site specificity in nucleotide-binding CRISPR-Cas proteins is governed by RNA molecules, rather than DNA-binding proteins, which can be more difficult to design and synthesize. Therefore, the CRISPR/Cas system offers advantages over zinc finger and transcription activator-like effector DNA-binding proteins. However, until now, genome editing methods using RNA-induced endonucleases (RGENs) based on the CRISPR/Cas system had not been developed. On the other hand, restriction fragment length polymorphism (RFLP), still widely used in molecular biology and genetics, is one of the oldest, simplest, and cheapest genotyping methods, but is often limited by the lack of suitable sites recognized by restriction endonucleases. Engineered nuclease-induced mutations (EMMs) are detected by various methods, including mismatch-sensitive T7 endonuclease I (T7E1) assays or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, dideoxy sequencing, and deep sequencing. While T7E1 and Surveyor assays are widely used, they are cumbersome. Furthermore, these enzymes tend to underestimate mutation frequencies because mutant sequences can form homozygous double-helixed structures with each other, making it impossible to distinguish between homozygous biallele mutant clones and wild-type cells. RFLP is preferred because it does not have these limitations. In fact, RFLP was one of the first methods for detecting engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction enzyme recognition sites. There may be no restriction enzyme recognition sites available at the target site of interest. Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (a) Schematic diagram of target DNA and chimeric RNA sequences. Red triangles indicate cleavage sites. PAM sequences recognized by Cas9 are shown in bold. Sequences in guide RNAs derived from crRNA and tracrRNA are shown in boxes and underlines, respectively.Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (b) In vitro cleavage of plasmid DNA by Cas9. A complete (intact) circular plasmid or ApaLI digested plasmid was incubated with Cas9 and guide RNA.Figure 2 shows Cas9-induced mutagenesis at episomal target sites. (a) Schematic diagram of a cell-based assay using an RFP-GFP reporter. GFP is not expressed by this reporter because the GFP sequence is frameshifted and fused with the RFP sequence. The RFP-GFP fusion protein is expressed only when the target site between the two sequences is cleaved by a site-specific nuclease.Figure 2 shows Cas9-induced mutagenesis at episomal target sites. (b) Flow cytometry of Cas9-transfected cells. The percentage of cells expressing the RFP-GFP fusion protein is shown.Figure 3 shows RGEN-induced mutations at endogenous chromosomal sites. (a) CCR5 locus. (Top) RGEN-induced mutations were detected using the T7E1 assay. Arrows indicate the predicted locations of DNA bands cleaved by T7E1. Mutation frequency (Indels (%)) was calculated by measuring band intensity. (Bottom) DNA sequences of wild-type (WT) and mutant clones of CCR5 and C4BPB. Regions of target sequences complementary to the guide RNA are enclosed in a box (boc). PAM sequences are shown in bold. Triangles indicate cleavage sites. Bases corresponding to microhomo