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System and method for genome editing

A genome editing and genome technology, applied in the field of genetic engineering, can solve problems such as genome editing of the AacC2c1 system that has not been proven

Active Publication Date: 2019-04-26
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, the AacC2c1 system has not been demonstrated to enable genome editing in eukaryotes

Method used

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  • System and method for genome editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148]Example 1. In vitro analysis of AaC2c1 nuclease activity.

[0149] First, the PAM sequence of the present invention from A. acidiphilus C2c1 was identified by in vitro nucleic acid cleavage. figure 1 A shows cleavage of AaC2c1 and sgRNAs targeting loci with various PAMs. The symbol "+" below the graph indicates strong in vitro cleavage activity. The results show that the PAM of AaC2c1 can be 5'TTTN-, 5'ATTN-, 5'GTTN-, 5'CTTN-, 5'TTC-, 5'TTG-, 5'TTA-, 5'TTT-, 5'TAN -, 5'TGN-, 5'TCN-, 5'ATC-.

[0150] Second, the temperature and acid-base tolerance of AaC2c1 were tested. figure 1 B shows the cleavage activity of AaC2c1 over a wide temperature range (4°C-100°C). figure 1 C Analysis of AaC2c1 cleavage activity at a wide range of pH values ​​(pH1.0-pH13.0). The symbol "+" below the graph indicates strong in vitro cleavage activity. The results show that AaC2c1 can work at 4°C-100°C, and the cutting efficiency is higher at about 30°C-60°C. AaC2c1 can work at pH1.0-pH12....

Embodiment 2Aa

[0158] Example 2 Genome Editing Activity of AaC2c1 in Mammalian Cells

[0159] In this example, the genome editing activity of AaC2c1 in mammalian cells was detected. The target sequences used are shown in Table 3 below.

[0160] image 3 A is a schematic diagram of the AaC2c1 sgRNA-DNA-targeting complex.

[0161] image 3 B shows T7EI analysis of indels generated at human RNF2 target sites. Numbers under lanes with mutations show Indel ratios. Triangles indicate cut fragments.

[0162] image 3 C shows from image 3 Sanger sequencing results of the cleavage products of B. Red font highlights PAM sequences.

[0163] T7EI experiments showed that AaC2c1 induced an indel at the mouse Nr1 locus ( image 3 D). image 3 E shows from image 3 Allelic sequence resulting from cleavage of target 1 of the Nrl gene of D.

[0164] Thus, AaC2c1 mediates robust genome editing in mammalian cells. Figure 4 The data further confirmed this conclusion. Figure 4 A AaC2c1 induces a...

Embodiment 3

[0170] Example 3, sgRNA optimization

[0171] This example optimizes the single guide RNA (sgRNA) that guides AaC2c1 genome editing. The original sgRNA was sgRNA1 constructed based on the tracrRNA in the AaC2c1 locus and the putative crRNA of A. acidoterrestris.

[0172] Image 6 A shows the structure of different versions of the sgRNA scaffold sequence with 5' truncated at stem-loop 3 of sgRNA1. Figure 5 A shows that truncation and disruption of stem-loop 3 of the sgRNA abolished the targeting activity of AaC2c1 to target site 8 of the human endogenous gene RNF2 in vivo. Image 6 B shows that truncation and disruption of stem-loop 3 of the sgRNA abolished AaC2c1 targeting activity in vitro. Image 6 C shows that truncation and disruption of stem-loop 3 of the sgRNA abrogates the targeting activity of AaC2c1 to target site 1 of the mouse endogenous gene Nrl in vivo.

[0173] Image 6 D shows the structure of different versions of the sgRNA scaffold sequence truncated and...

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Abstract

Provided are a genome editing system CRISPR-C2c1 for site-directed PEGylation of target sequences in cell genomes and uses thereof. The system comprises a C2c1 protein or a variant thereof and a guideRNA. Also provided are a method for site-directed PEGylation of target sequence in cell genome by the genome editing system CRISPR-C2c1, and a pharmaceutical composition containing the genome editingsystem CRISPR-C2c1.

Description

technical field [0001] The invention relates to the field of genetic engineering. In particular, the present invention relates to novel genome editing systems and methods. More specifically, the present invention relates to a new CRISPR-C2c1 system capable of efficiently editing the genome of a cell and its use. Background technique [0002] CRISPR (Clustered regularly interspaced short palindromic repeats, clustered regularly interspaced short palindromic repeats) system is an immune system produced by bacteria during evolution to defend against foreign gene invasion. Among them, the type II CRISPR-Cas9 system is a system in which a Cas9 protein is mediated by two small RNAs (crRNA and tracrRNA) or a synthetic small RNA (sgRNA) for DNA cleavage. Type III) The simplest of the CRISPR systems. Because the system is simple and easy to operate, it was modified in 2013 and successfully realized the editing of eukaryotic genomes. The CRISPR / Cas9 system has quickly become the h...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/85C12N15/90A61K48/00A61K38/46A61P35/00A61P29/00A61P25/16A61P9/00A61P25/28A61P25/30A61P25/18A61P25/00A61P27/02
CPCA61K38/465A61K48/005A61P9/00A61P25/00A61P25/16A61P25/18A61P25/28A61P25/30A61P27/02A61P29/00A61P35/00C12N15/8213C12N15/85C12N15/902C12N15/907C12N2810/10C12N2800/106C12N2800/107C12N2310/20C12N15/113C12N15/1138C12N9/22C12N15/11
Inventor 李伟周琪滕飞
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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