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Method for reducing CRISPR/Cas9 mediated embryo gene editing missing rate

An embryonic gene and editing technology, applied in the field of off-target rate of embryonic gene editing, can solve problems such as insufficiency, limited efficiency and accuracy, and off-target, and achieve the effects of wide application, easy manipulation, and reduced off-target mutation rate

Inactive Publication Date: 2018-01-09
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, research on Cas9 has only been done in three prokaryotic fertilized eggs that are abnormally fertilized and cannot develop into viable embryos, but it is found that the CRISPR / Cas9 system has limitations in efficiency and accuracy, especially in frequent off-targets in research Phenomenon
Although there are many ways to try to improve the high-frequency off-target phenomenon of CRISPR / Cas9 gene editing technology, various mutational defects brought about by it are still unavoidable

Method used

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  • Method for reducing CRISPR/Cas9 mediated embryo gene editing missing rate
  • Method for reducing CRISPR/Cas9 mediated embryo gene editing missing rate
  • Method for reducing CRISPR/Cas9 mediated embryo gene editing missing rate

Examples

Experimental program
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Effect test

Embodiment 1

[0033] In this example 1, discarded eggs that cannot be fertilized normally in conventional IVF were selected, and ICSI method was used to promote the fertilization of these eggs and develop into diploid embryos. We have confirmed that the combined injection of intracytoplasmic sperm injection in discarded eggs can Efficient sgRNA-E with Cas9 mRNA targeting human encoding recombination-activated gene 1 (RAG1). Subsequently, the T7EN1 cleavage method and deep sequencing were used to detect the targeting effect and off-target efficiency of Cas9 / sgRNA technology in gene editing of human diploid embryos.

[0034] 1.1 Using the method provided by the present invention, using the CRISPR / Cas9 system to perform gene editing on human diploid embryos derived from discarded eggs:

[0035] 1) Sources of discarded human eggs:

[0036] All couples of infertile patients signed the informed consent form for the donation of discarded eggs due to unfertilized eggs during the in vitro fertiliza...

Embodiment 2

[0077] Reducing off-target damage using a Cas9 nickase / dual sgRNA strategy

[0078] In this example, an editing strategy requiring the simultaneous application of sgRNAs at 2 adjacent positions was attempted to be applied to human diploid embryos. We chose sgRNA-E and sgRNA-1, which are 11bp apart. Four (#84-87) and three (#95-97) human diploid embryos were collected from two independent experiments. Whole-genome DNA was amplified by PCR and used for T7EN1 digestion analysis, and restriction bands (#84-87, #95) were found in 5 samples ( Figure 9 &10 and Table 2). This result is consistent with previous studies: the dual sgRNA strategy has a higher targeting efficiency than the single sgRNA strategy (11 / 29vs 5 / 7) (Table 2). The results of direct sequencing of PCR products showed that the TA clone product had multiple peaks, further confirming this result ( Figure 11 &table 3). Likewise, targeting efficiencies ranging from undetectable 0% to 100% (#84) also indicated that...

Embodiment 3

[0083] The CRISPR / Cas9 system mediates complex genome engineering in human embryos

[0084] To verify whether the CRISPR / Cas9 system can mediate multiple gene editing in human embryos, we designed two sgRNAs targeting the UBE3A gene (encoding ubiquitin protein ligase E3A), namely UBE3A-sgRNA-1 and 2, together with RAG1- sgRNA-E and 1 were simultaneously microinjected into human embryos. Four (#147-150) and six (#151-156) embryos were collected from two independent experiments. All 4 embryos in the first experiment successfully completed whole-genome amplification, and 4 out of 6 embryos in the second experiment successfully completed whole-genome amplification, and the amplified DNA was subjected to T7EN1 digestion experiment. The results show( Figure 15 &16): Of the 8 embryos, 5 (#147-150, 152) had RAG1-targeted restriction bands, and 3 (#149-150, 152) had UBE3A-targeted restriction bands. It is worth noting that 3 out of 8 embryos (#149-150,152) had both RAG1 and UBE3A m...

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Abstract

The invention relates to a method for reducing CRISPR / Cas9 mediated embryo gene editing missing rate. The method comprises the following steps: compounding a sgRNA sequence of a targeting gene and constructing a sgRNA expression plasmid carrying T7 promoter; in vitro transcribing Cas9mRNA and sgRNA; and importing intracytoplasmic sperm into unfertilized MII ovum by adopting intracytoplasmic sperminjection (ICSI) and meanwhile importing Cas9mRNA and sgRNA. According to the method provided by the invention, the targeting editing efficiency is high and the missing mutation rate is low.

Description

technical field [0001] The present invention relates to gene editing, in particular how to reduce the off-target rate of CRISPR / Cas9-mediated gene editing in embryos. Background technique [0002] With the advent of a new era of genetics and genomics, advanced DNA sequencing technologies have uncovered the molecular code that regulates ontogeny abnormalities. Gene editing, once known as gene targeting technology, mainly relies on the specific principle of complementary base pairing to modify the genomic DNA sequence. Currently, most genome editing is achieved with the help of custom endonucleases, including zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and the CRISPR-Cas9 system. ZFNs and TALENs technologies require complex protein assembly engineering, while the CRISPR-Cas9 system can be easily completed by replacing the guide RNA. Therefore, as an efficient, easy-to-design, easy-to-use, and multiple-target editing gene editing t...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/873
Inventor 孙海翔李朝军黄行许颜桂军周建奎
Owner NANJING DRUM TOWER HOSPITAL
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