Homologous recombination mechanism-mediated precise sequence replacement gene editing method and its component structure

A technology of homologous recombination and gene editing, applied in the field of gene editing, can solve the problems of no off-target risk, dependence, high off-target risk, etc., and achieve the effect of increasing the probability of events

Active Publication Date: 2022-08-02
ANHUI AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is the dependence on the PAM sequence; the other is the high risk of off-target; the third is that there are few gene editing technology tools for precise sequence replacement through homologous recombination, which do not rely on PAM, and have no or very low off-target risk

Method used

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  • Homologous recombination mechanism-mediated precise sequence replacement gene editing method and its component structure
  • Homologous recombination mechanism-mediated precise sequence replacement gene editing method and its component structure
  • Homologous recombination mechanism-mediated precise sequence replacement gene editing method and its component structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] In this example, taking the E. coli gene aroA target segment as an example, how to use the PCRRISR structure and the donor template of the present invention for sequence replacement is described in detail.

[0041] (1) First, prepare a donor template, and the way to obtain the donor template is:

[0042] (1.1) Determine the sequence of the target segment;

[0043] In this example, the E. coli gene aroA was used as the target segment, and its sequence was replaced.

[0044] (1.2) Insert or delete 5-6 donor sites in the target segment sequence to form a donor template. In this example, a MsDFID donor template is used. The MsDFID donor template contains 6 donor sites, 5 of which are DNA fragment insertions, 1 site is DNA fragment deletion, and the length of the homology arm is 60 bp. See image 3 Part (A) of . Preferably, the longest DNA insertion segment is 65 bp, and the longest DNA deletion segment is 35 bp.

[0045] (2) Connect the PCRRISR "hairpin" structure at th...

Embodiment 2

[0060] In this example, the inventors used the NJZL plasmid as the donor template, combined with the PCRRISR "hairpin" structure, to realize the sequence replacement of the target site of the transposon in the E. coli genome.

[0061] (1) First, prepare a donor template, and the way to obtain the donor template is:

[0062] (1.1) Determine the sequence of the target segment;

[0063] In this example, one transposon sequence of Escherichia coli (GenBank No. CP026085.1 (gene product="IS4 family transposase ISVsa5") was used as the target segment to replace its sequence.

[0064] (1.2) Insert or delete several donor sites in the target segment sequence to form a donor template. In this example, the NJZL plasmid donor template is used, and the donor template contains 5 donor sites, see Figure 4 Part (A) of .

[0065] (2) Connect the PCRRISR "issuer" structure to the front end of the donor template

[0066] After inserting multiple donor sites into the target segment sequence,...

Embodiment 3

[0074] In a similar manner to Example 1 and Example 2, in this example, the dctA target segment of the E. coli gene was replaced by sequence, and the replacement results were as follows Figure 5 shown.

[0075] Figure 5 Shown is a diagram of the experimental procedure for the generation of sequence replacement in the dctA target segment of the E. coli gene using a series of vectors comprising a MsDFID donor template fused to a PCRISR construct.

[0076] Figure 5 (A) shows the structure of the donor vector and the electropherogram of the genotyping PCR product obtained by transforming E. coli genomic DNA with these vectors and amplifying it, wherein, in the figure, 501 represents the PCR primer for identifying the genotype of the target segment ; 502 represents the fusion structure of the MsDFID donor template and PCRISR in the donor vector CY7869-6; 503 represents the target segment and its flanking sequence in the E. coli gene dctA; 504 represents the genotype identifica...

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Abstract

The present invention discloses a method and element structure for precise sequence replacement gene editing mediated by homologous recombination mechanism. The MsDFID sequence, which simultaneously serves as a guide RNA and a donor template, is also referred to as a MsDFID guide and a donor RNA; (2) the 3' end of the MsDFID donor template is linked into a clustered and regularly spaced short repeat DNA palindromic structure, namely PCRRISR, or Cas9 guide RNA backbone, or E. coli CRISPR-Cas3 palindromic repeats. The latter three sequences are used as the guide RNA backbone; (3) the above-mentioned fusion sequence is used to construct a donor vector, transform Escherichia coli, and the insertion or deletion of the DNA fragment contained in the donor site in the MsDFID donor template is replaced by the corresponding position of the target segment. point. The invention successfully realizes gene editing with sequence replacement as the only effect, and solves several main problems faced by the current gene editing system: dependence on PAM sequences, high off-target risk and difficulty in realizing precise sequence replacement.

Description

technical field [0001] The invention relates to the field of gene editing, in particular to a brand-new PAM-independent, homologous recombination mechanism-mediated precise sequence replacement gene editing method. Background technique [0002] Gene editing is an emerging and relatively precise genetic engineering technology or process that can modify specific target genes in the genome of an organism. Gene editing relies on genetically engineered nucleases, also known as "molecular scissors," to create site-specific double-strand breaks (DSBs) at specific locations in the genome, inducing organisms to undergo non-homologous end joining (NHEJ) or homologous end joining. Source recombination (HR) to repair DSBs because this repair process is error-prone, leading to targeted mutations. This targeted mutation is gene editing. [0003] Currently, in the field of gene editing, a very common and effective editing technology is CRISPR-Cas9 technology. CRISPR is a defining featur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/70C12N15/90
CPCC12N15/113C12N9/22C12N15/70C12N15/902C12N2310/20C12N2310/531
Inventor 许永汉林新春马传喜李金才胡晓渝王晓波武德传齐泽宇吕蔺蒋欣瑜桂昊
Owner ANHUI AGRICULTURAL UNIVERSITY
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