Effective knockout of same gene achieved by co-injection of multiple sgRNAs

A gene knockout and gene technology, applied in genetic engineering, using microinjection method, recombinant DNA technology, etc., can solve the problems of gene acquisition, frame shift mutation, imprecise gene editing, etc.

Active Publication Date: 2019-01-18
肇庆华夏凯奇生物技术有限公司
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AI Technical Summary

Problems solved by technology

[0003] CRISPR / Cas9 system-mediated specific gene knockout (knockout) is the use of sgRNA (single guidedRNA) to guide the Cas9 protein to cut double-stranded DNA through target sequence complementarity, resulting in non-homologous end joining (NHEJ) Repair, resulting in frameshift mutation (frameshift mutation), leading to gene knockout, this method mainly has the following disadvantages in practical application: First, the mechanism of non-homologous end joining is prone to random insertion and deletion (indel), so that near the breakpoint Potentially introduces new bases randomly, leading to imprecise gene editing
However, due to the limitation of BE3 editing efficiency and other factors, there are no examples of effective gene knockout obtained by this method.

Method used

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  • Effective knockout of same gene achieved by co-injection of multiple sgRNAs
  • Effective knockout of same gene achieved by co-injection of multiple sgRNAs
  • Effective knockout of same gene achieved by co-injection of multiple sgRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] BE3-mediated base editing was performed on cell lines, and stop codons were introduced to achieve gene knockout. Carry out gene knockout of cell lines (by electroporation or liposome transfection) according to routine operation, taking liposome transfection as an example.

[0038] (1) Taking N2a cells as an example, the present invention carries out culture and transfection of eukaryotic cells: N2a cells are inoculated and cultured in DMEM high-sugar culture solution (HyClone, SH30022.01B) supplemented with 10% FBS, which contains penicillin ( 100U / ml) and streptomycin (100μg / ml).

[0039] (2) Divide into 6-well plates before transfection, and perform transfection when the density reaches 70%-80%.

[0040] (3) Transfection Take liposome transfection as an example. Follow Lipofectamine TM 2000 Transfection Reagent (Invitrogen, 11668-019) operating manual, taking SpCas9nickase as an example, mix 2 μg BE3 plasmid and 1 μg pGL3-U6-EGFP-sgRNA plasmid, co-transfect into ea...

Embodiment 2

[0079] BE3-mediated base editing was performed on mouse embryos, and a stop codon was introduced to achieve gene knockout.

[0080] (1) In vitro transcription: BE3 plasmid was linearized with AgeI enzyme (NEB, R3552L), and transcribed in vitro with T7ULTRA (Ambion, AM1345). BE3 mRNA was purified using RNeasy Mini Kit (QIAGEN, 74104). The sgRNA nucleotides were annealed into the pUC57 sgRNA expression vector with T7 promoter (Addgene, 51132). The sgRNAs were then amplified and transcribed using the MEGAshorttranscript T7 kit (Ambion, AM1345). sgRNA was purified with MEGAclear kit (Ambion, AM1908) and recovered by ethanol precipitation.

[0081] (2) Microinjection: Female C57BL / 6J mice treated with superovulation were mated with male C57BL / 6J mice, and fertilized eggs were collected from the oviduct on day 0.5. Fertilized eggs were injected with BE3mRNA (50ng / μl) and specific sgRNA (25ng / μl). The injections are as follows:

[0082] 1. Pcdc1

[0083] Combination 1: BE3mRNA+...

Embodiment 3

[0113] Construction of BE3-mediated knockout mice.

[0114] Mouse embryo collection, microinjection, embryo culture and embryo transfer were performed according to routine operations.

[0115] (1) Microinjection: Fertilized eggs were injected with BE3 mRNA and specific sgRNA (Experiment 1: PD1-sg1+2+3, Tyr-sg1+2), or BE3 mRNA and specific sgRNA (Experiment 2: PD1-sg1+2 +3, Tyr-sg1+2+3). Routine embryo transfer;

[0116] (2) Genotype analysis:

[0117] A. Genomic DNA was extracted by pruning the tail of conventional mice: after cleavage and digestion with 100 μg / ml proteinase K in the lysate (10 μM Tris-HCl, 0.4M NaCl, 2 μM EDTA, 1% SDS), dissolve to 50 μl after phenol-chloroform extraction deionized water.

[0118] B. Use a pair of primers N-For and N-Rev to carry out PCR amplification, and use AxyPrep PCR cleanup kit (AXYGEN, AP-PCR-250G) to purify and obtain PCR recovered products. The PCR reaction system is:

[0119] 300-400ng genomic DNA

[0120] 25μl 2X Buffer

[0...

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Abstract

Effective knockout of the same gene achieved by co-injection of multiple sgRNAs. The knockout method comprises the following steps: selecting at least two target sequences of a coding region of a geneto be knocked out so as to respectively contain an intact target codon CAA, CAG or CGA; At least two sgRNA sequences are utilized to map BE3 to the corresponding target sequence so that the target single base C in the target codon becomes T so that the termination codon TAA or TAG, TGA is introduced correspondingly to realize gene knockout; At least two sgRNA sequence are sequences complementaryto that at least two target sequence, respectively, wherein the at least two sgRNAs are co-injected to effect effective knockout of the same gene. The present invention provides a gene knockout methodwhich is more efficient and has high accuracy and low miss-targeting effect.

Description

technical field [0001] The invention relates to the improvement and application of base editing-based gene knockout method strategy. Background technique [0002] Gene editing technology refers to the process of "editing" the target gene and introducing desired changes into the target site of genomic DNA. Early gene editing was achieved through the discovery of endogenous homologous recombination. However, traditional eukaryotic targeted gene manipulation has disadvantages such as low gene targeting efficiency and limited application range. The discovery and application of type II CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas9 endonuclease) system has broken the original limitations, and this system has been proved to be a tool for multipurpose gene editing [Le et al., 2013; Patrick et al .,2014]. [0003] CRISPR / Cas9 system-mediated specific gene knockout (knockout) is the use of sgRNA (single guidedRNA) to guide the Cas9 protein t...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/89C12N9/22
CPCA01K67/0276A01K2227/105C07K14/47C12N15/8509C12N2310/20
Inventor 黄行许王新贾堃
Owner 肇庆华夏凯奇生物技术有限公司
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