Analysis method of genomic DNA (deoxyribonucleic acid) fragment editing precision applicable to CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, and application

An analysis method and genome technology, applied in the field of analysis of the accuracy of genome DNA fragment editing, to achieve the effect of improving experimental efficiency

Active Publication Date: 2017-08-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the existing technology, a large number of complicated experiments are required to use the CRISPR / Cas9 system for high-precision editing of genomic DNA fragments

Method used

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  • Analysis method of genomic DNA (deoxyribonucleic acid) fragment editing precision applicable to CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, and application
  • Analysis method of genomic DNA (deoxyribonucleic acid) fragment editing precision applicable to CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, and application
  • Analysis method of genomic DNA (deoxyribonucleic acid) fragment editing precision applicable to CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Example 1 Transfection of sgRNAs targeting the CtIP gene can improve the precise ligation efficiency after deletion of DNA fragments

[0187] 1. Construction of sgRNAs plasmids for STM sites and CtIP genes

[0188] (1) Purchase primers

[0189] Purchase forward and reverse deoxyoligos with 5' hanging ends "ACCG" and "AAAC" that can be complementary paired for the STM site (β-globin RE1) and the sgRNAs targeting sequence of the CtIP gene from Shanghai Sunny Biotechnology Co., Ltd. Nucleotides.

[0190] Forward and reverse deoxy oligonucleotides:

[0191] β-globin RE1sgRNA1F: accgATTGTTGTTGCCTTGGAGTG (SEQ ID NO.1)

[0192] β-globin RE1sgRNA1R: aaacCACTCCAAGGCAACAACAAT (SEQ ID NO.2)

[0193] β-globin RE1sgRNA2F: accgCTGGTCCCCTGGTAACCTGG (SEQ ID NO.3)

[0194] β-globin RE1sgRNA2R: aaacCCAGGTTACCAGGGGACCAG (SEQ ID NO.4)

[0195] CtIPsgRNA1F: accgGAGCAGAGCAGCGGGGCAA (SEQ ID NO.5)

[0196] CtIPsgRNA1R: aaacTTGCCCCGCTGCTCTGCTC (SEQ ID NO. 6)

[0197] CtIPsgRNA2F: accgTT...

Embodiment 2

[0267] Example 2 CtIP mutations in cell lines can effectively improve the precision ligation efficiency of deletion of target DNA fragments

[0268] 1. Obtain CtIP-mutated cell lines by CRISPR system

[0269] 1) HEK293T cells were cultured in a culture flask, and when they grew to 80-90% of the culture flask, the grown cells were plated in a 12-well plate with DMEM complete antibiotic-free medium, and cultured overnight. When the cells in the 12-well plate grew to 80-90%, the prepared humanized Cas9 plasmid (800ng) and the sgRNAs plasmid (600ng each) at the CtIP site were transfected with Lipofectamine2000.

[0270] 2) Add Puromycin (2μg / ml) to the cells 48 hours after transfection for four days of drug screening, then culture them in fresh medium for eight days, collect the cells, count the cells evenly dispersed, and then dilute to a certain number of species Into a 96-well plate (only one cell per well), after 6 days of culture, the well plate with only one cell cluster co...

Embodiment 3

[0287] Example 3 3-AP Improves the Precision Ligation Efficiency of DNA Fragment Deletion

[0288] 1. Cell line transfection with Lipofectamine 2000 at the STM site

[0289] HEK293T cells and CtIP mutant cells were plated in 12-well plates with DMEM complete anti-antibody medium and cultured overnight. When the cells in the 12-well plate grew to 80-90%, the medium was removed, and DMEM containing DMSO or different concentrations of 0.2 μM, 0.4 μM, 0.8 μM, and 1.6 μM 3-AP (SML0568, Sigma) was added without Anti-medium, the prepared humanized Cas9 plasmid (800ng) and sgRNAs (600ng each) targeting the STM site were transfected with Lipofectamine 2000. After 24 hours, remove the medium, add DMEM complete double antibody medium (add 10% fetal bovine serum and 1% penicillin double antibody), and after another 24 hours, collect the cells, use the genome extraction kit ( Genomic DNA Purificationkit, Promega) was used to extract the genome, and each sample had two replicates.

[0...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an analysis method of genomic DNA (deoxyribonucleic acid) fragment editing precision applicable to a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease 9) system, and application of the analysis method. The analysis method is characterized in that a cutting manner of Cas9 nuclease to genomic DNA double strands is divided into blunt end cutting and protruding end cutting; a cutting end ratio corresponding to a blunt end cutting manner is a blunt broken end ratio; a cutting end ratio corresponding to a protruding end cutting manner is a protruding broken end ratio; and editing precision of candidate sgRNA (small guide ribonucleic acid) combinations and the selected Cas9 nuclease to genomic DNA fragments is predicted by predicting corresponding broken end sequences of the candidate sgRNA combinations in each cutting manner and combining the blunt broken end ratio and the protruding broken end ratio. With the adoption of the analysis method, precision prediction can be first performed on an editing method and a complicated experiment can be saved, so that the experiment efficiency is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an analysis method for editing accuracy of genomic DNA fragments applicable to a CRISPR / Cas9 system and an application thereof. Background technique [0002] Biotechnology is crucial to the country's bio-industrial development as well as agriculture and health industries. Since the completion of the Human Genome Project and the Encyclopedia of DNA Elements, scientists have analyzed and identified a large number of genes and DNA regulatory elements in the genome [1,2]. DNA regulatory elements that play an important role in the regulation of gene expression include promoters, enhancers, silencers, and insulators. However, the functions of most regulatory elements have not been experimentally verified and elucidated [2-8]. Exploring the function of genes and DNA regulatory elements can be studied through genetic DNA segment editing. [0003] Early gene editing and gene fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44C12N15/85
CPCC12N9/22C12N15/85C12N2800/107C12N2810/10C12Q1/44C12Q1/68G01N2333/922C12Q2521/301
Inventor 吴强李金环寿佳
Owner SHANGHAI JIAO TONG UNIV
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