Improved gene editing system

A gene editing and genome technology, applied in the field of genetic engineering, can solve problems such as inaccurate mutation types, reduced efficiency, and unpredictability

Pending Publication Date: 2022-02-01
SUZHOU QI BIODESIGN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

et al. (2017) used Cas9 fusion 3' repair exonuclease 2 (Trex2), which significantly increased the frequency of deletion mutations and the deletion fragments were longer, but the type of mutation was still imprecise and unpredictable; using a pair of sgRNAs for targeting Deletion can obtain specific long fragment deletions, but at the same time it will also produce inversions, small fragments InDel, etc., which also greatly reduces the efficiency of the former ( et al., 2017)
In order to obtain accurate fragment deletions, Wolfs et al. (2016) fused Cas9 with TevI nuclease, which recognized the enzyme cleavage site and cut double-stranded DNA. Restriction of restriction sites results in lower efficiency of the system
So far, no tool has been developed that can perform efficient, accurate and predictable deletion of short fragments within the range of Protospacer

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1. Construction of a gene editing system (ACD) for precise short fragment deletion

[0140] A single base editing system has been established in 2016 (Komor et al., 2016; Ma et al., 2016; Nishida et al., 2016). The system uses nCas9 (D10A) to guide cytosine deaminase to act on the non-complementary strand of the DNA target site, and deaminates cytosine (C) in a specific region to uracil (U), uracil (U) During DNA replication, it will be replaced by thymine (T), thus achieving precise single base substitution of C-to-T. In the repair process of animal and plant organisms, uracil-DNA glycosylase (Uracil-DNA Glycocasylase, UDG) will preferentially recognize the U base and remove the N-glycosidic bond of the base to form an apurinic or apyrimidinic site (apurinic or apyrimidinic site, AP site), and then under the action of AP lyase (AP lyase), the U base is repaired into the original C base by base excision repair. Therefore, Uracil-DNA Glycocasylase inhibitor (UG...

Embodiment 2

[0143] Example 2, the analysis of the missing type produced by the ACD system

[0144] Sequence analysis of the Deletion mutations generated by the ACD system on different target sites ( Figure 3-8 ), except for a few types, most of the mutation types are in line with expectations, all of which are APOBEC3A action bases (NGG (PAM) corresponds to C bases; CCN (PAM) corresponds to G bases) to the Cas9 cleavage site Deletion between points. However, due to the asymmetry of Cas9 in cutting the double strand, Cas9 will cut between the 3-4 or 4-5 near the PAM end. In addition, the bases that APOBEC3A acts on on the non-target strand will use the target strand as a template to form 1-2 bases that pair with the complementary strand during the repair process. Therefore, 1-2 bases that are compatible with the target strand may also be introduced bases paired with complementary strands.

[0145] The efficiency of the ACD system to generate Insertion is very low, but the efficiency of...

Embodiment 3

[0146] Embodiment 3, construct AFID (APOBEC-Cas9 Fusion-Induced Deletion) system

[0147] The present invention selects human APOBEC3A with high deamination activity and wide deamination window to construct the AFID-3 system, and screens a higher deamination activity and narrow window APOBEC3Bctd to replace APOBEC3A to construct the eAFID-3 system ( Figure 9 and Figure 10 ). A comparative analysis of the deletion efficiencies of Cas9, AFID-3 and eAFID-3 on the endogenous gene targets of rice and wheat showed that the efficiency of deletion mutations was significantly increased compared with Cas9, AFID-3 and eAFID-3, and its The average deletion mutation rate is 2.2 times and 2.6 times that of Cas9, which fully demonstrates the high efficiency of the AFID system.

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Abstract

Provided is a gene editing system for editing a target sequence in the genome of a cell, comprising a CRISPR nuclease, a cytosine deaminase, an AP lyase, a guide RNA and optionally an uracil-DNA glycosylase. Also provided are a method of producing a genetically modified cell, and a kit comprising the gene editing system.

Description

technical field [0001] The invention relates to the field of genetic engineering. In particular, the present invention relates to an improved gene editing system. More specifically, the present invention relates to a gene editing system capable of accurately editing eukaryotic cell genomes, especially predictable and accurate deletion of polynucleotides. [0002] Background of the invention [0003] In recent years, with the continuous development of genome editing technology, a large number of gene editing tools have been developed, improved and applied, including gene knockout tools mediated by SpCas9 to gene knockout tools mediated by nCas9(D10A) fusion cytosine deaminase (Cytosine deaminase). Guided single base editing tools, etc. Guided by the guide RNA, SpCas9 binds and cuts double-stranded DNA to form a double-strand break (Double-stranded break, DSB), which often introduces insertions and / or deletions of different fragment lengths during the repair process of the bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/85C12N9/22
CPCC12N9/22C12N15/8213C12Y305/04001C12N9/78C12N9/2497C12N9/88C12Y402/99018C12N15/90C12N2310/20C12N15/79
Inventor 高彩霞张华伟王升星
Owner SUZHOU QI BIODESIGN BIOTECHNOLOGY CO LTD
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