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Reagent and method for repairing E8SJM<-1G>A> mutation related to cholesteryl ester storage disease by means of base editing

A base editing and kit technology, applied in the field of E8SJM-1G>A reagents, which can solve the problems of fragmentation, imprecise gene editing, low HDR efficiency, etc.

Active Publication Date: 2019-04-23
国家卫生健康委科学技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CRISPR / Cas9-mediated gene editing is when sgRNA (single guided RNA) guides Cas9 protein to position and cut double-stranded DNA through target sequence complementarity, resulting in double-strand breaks (DSB), under the condition of no template , non-homologous end joining repair occurs, resulting in frameshift mutation (frameshift mutation), leading to gene knockout (knockout); under the condition of template, it is repaired by homologous recombination to realize gene knockin (knockin), due to HDR Inefficient (integration rarely occurs) and non-homologous end-joining mechanisms are prone to random insertions and deletions (indels), making it possible to randomly introduce new bases near breakpoints, leading to imprecise gene editing

Method used

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  • Reagent and method for repairing E8SJM&lt;-1G&gt;A&gt; mutation related to cholesteryl ester storage disease by means of base editing
  • Reagent and method for repairing E8SJM&lt;-1G&gt;A&gt; mutation related to cholesteryl ester storage disease by means of base editing
  • Reagent and method for repairing E8SJM&lt;-1G&gt;A&gt; mutation related to cholesteryl ester storage disease by means of base editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] In this example, Cas9 / sgRNA combined with ssODN was used to make mutations on the cell line E8SJM -1G＞A For mutant cell lines, this method will utilize the RNA binding ssODN form of Cas9mRNA and sgRNA to realize ( Figures 1A-1D ), the Cas9 used may be spCas9 (SEQ ID NO.4, addgene: #44758) or XCas9 (SEQ ID NO.5, addgene: 108379).

[0039] 1.1 Plasmid construction

[0040] Near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.2) and synthesize oligos. The upstream sequence is: 5'-taggATGTTACACTGGAGCCAGGT-3' (SEQ ID NO.(6)), and the downstream sequence is: 5'- aaacACCTGGCTCCAGTGTAACAT-3' (SEQ ID NO. (7)), the upstream and downstream sequences passed the program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) and ligated to the PUC57-T7 sgRNA carrier (addgene: 51132) linearized with BsaI (NEB: R0539L). The linearization system is as follows: PUC57-T7sgRNA 2μg; buffer (NEB: R0539L) 6μL; BsaI 2μL; ddH 2 O to make up to 60 μL. Digest overnight at...

Embodiment 2

[0058] In this example, for the obtained homozygous mutant cell line, the E8SJM -1G＞A Implement the fix. In this embodiment, spCas9-ABE and XCas9-ABE two mRNAs combined with corresponding repair sgRNA are used to repair the mutation site ( Figure 2A-Figure 2D , Figure 3A and Figure 3B ).

[0059] 1.1 Plasmid construction

[0060] Near the mutation site, design and repair re-sgRNA, synthesize oligos, the upstream sequence is: 5'-taggCCAaGTAGGCATTCCAGGAG-3' (SEQ ID NO. (6)), the downstream sequence is: 5'-aaacCTCCTGGAATGCCTACTTGG-3' (SEQ ID NO.(7)), the upstream and downstream sequences are annealed through the program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) annealing, ligation onto the PUC57-T7 sgRNA vector (addgene: 51132) linearized with BsaI (NEB: R0539L). The linearization system is as follows: PUC57-T7sgRNA 2μg; buffer (NEB: R0539L) 6μL; BsaI 2μL; ddH 2 O to make up to 60 μL. Digest overnight at 37°C. The ligation system is as follow...

Embodiment 3

[0077] In this example, for the obtained homozygous mutant cell line, the E8SJM -1G＞A Implement the fix. In this embodiment, the protein of XABE will be used in conjunction with the corresponding repair sgRNA to repair the mutation site ( Figure 2C , Figure 2D ).

[0078] 1.1 Plasmid construction

[0079] Near the mutation site, design and repair re-sgRNA, synthesize oligos, the upstream sequence is: 5'-taggCCAaGTAGGCATTCCAGGAG-3' (SEQ ID NO. (6)), the downstream sequence is: 5'-aaacCTCCTGGAATGCCTACTTGG-3' (SEQ ID NO.(7)), the upstream and downstream sequences are annealed through the program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) annealing, ligation onto the PUC57-T7 sgRNA vector (addgene: 51132) linearized with BsaI (NEB: R0539L). The linearization system is as follows: PUC57-T7sgRNA 2μg; buffer (NEB: R0539L) 6μL; BsaI 2μL; ddH 2 O to make up to 60 μL. Digest overnight at 37°C. The ligation system is as follows: T4 ligation buffer (NEB:...

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Abstract

The invention provides a reagent and method for repairing E8SJM<-1G>A> mutation related to a cholesteryl ester storage disease by means of base editing. A kit efficiently repairing the E8SJM<-1G>A> mutation comprises a base editing system and repair re-sgRNA aiming at an E8SJM<-1G>A> site. Accordingly, by means of a base editing technology and accurate AG single base mutation, the E8SJM<-1G>A> mutation can be repaired, and an efficient and safe method for treating the cholesteryl ester storage disease caused by the mutation is provided.

Description

technical field [0001] The invention relates to the field of gene repair, more specifically, the use of base editing to repair E8SJM associated with cholesteryl ester storage disease -1G＞A reagents and methods. Background technique [0002] Cholesterol ester storage disease (CESD) is an autosomal recessive genetic disease. The disease is due to loss of lysosomal acid lipase (LAL) activity. The lack of LAL causes cholesterol esters and triglycerides to be stored in lysosomes in liver, spleen and other tissues, leading to hepatosplenomegaly 1 . The clinical symptoms of cholesteryl ester storage disease appear at about one year old, continue until adulthood, and eventually die due to liver failure. The worldwide incidence of the disease is extremely low, possibly due to a proportion of misdiagnosed or missed diagnoses 2 . Loss of LAL activity is caused by mutations in the gene encoding it, LIPA. More than 30 related mutations have been found in reported CESD patients 2 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90
CPCC12N15/113C12N15/85C12N15/907C12N2310/20C12N2310/10
Inventor 马旭李广磊金孝华党露
Owner 国家卫生健康委科学技术研究所