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Method for evaluating CRISPR/Cas9 gene editing efficiency or miss frequency

A gene editing, off-target technology, applied in the field of bioengineering, can solve the problems of time-consuming, labor-intensive, low sensitivity, and inapplicability

Active Publication Date: 2019-06-11
ZHEJIANG FORESTRY UNIVERSITY
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  • Abstract
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Problems solved by technology

However, these methods are often time-consuming and labor-intensive, and even insensitive
The methods used to detect whether CRISPR / Cas9 is edited include mismatch enzyme method, high-throughput sequencing method, molecular identification, high-resolution melting curve, etc. These methods have certain shortcomings and are not suitable for large-scale screening

Method used

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  • Method for evaluating CRISPR/Cas9 gene editing efficiency or miss frequency
  • Method for evaluating CRISPR/Cas9 gene editing efficiency or miss frequency
  • Method for evaluating CRISPR/Cas9 gene editing efficiency or miss frequency

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Experimental program
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Embodiment

[0045] 1. Construction of CRISPR / Cas9 vector

[0046] 1.1 Cloning the reporter gene into the CRISPR / Cas9 vector

[0047] Using Arabidopsis genomic DNA as a template, ZYP11-FP (such as SEQ ID NO: 17) and ZYP11-BP (such as SEQ ID NO: 18) as primers, amplify the seed-specific expression of the At2S3 gene promoter to drive mCherry fluorescent protein gene; after electrophoresis detection has the At2S3 band, the first PCR product is diluted 100 times, and the PCR product after dilution is used as a template, ZYP12-FP (such as SEQ ID NO: 19) and ZYP12-RP (such as SEQ ID NO: 19) and ZYP12-RP (such as SEQ ID NO: 19) ID NO: 20) was used as the primer for the second amplification, and the restriction sites BamHI and EcoRI were cloned to both ends of the At2S3 fragment. The PCR product was BamHI-EcoRI-At2S3-BamHI, and its length was 455bp. The amplified The product was recovered from the gel for future use.

[0048] Digest the pFGC-35S-mCherry-NOS plasmid vector with BamHI (plasmid map...

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Abstract

The invention relates to a method for evaluating CRISPR / Cas9 gene editing efficiency or miss frequency, and belongs to the field of bioengineering. The method mainly comprises the following three parts: firstly, selecting a marker gene mCherry to be inserted into an expression vector in order to quickly screen a transgenic plant; secondly, in order to quickly identify the editing efficiency and the miss frequency of the CRISPR / Cas9 gene, inserting fatty acid dehydrogenase FAD2 and FAD3 as target genes into a vector; and finally, utilizing the change of fatty acid components after target gene mutation, and by detecting the composition of C18 unsaturated fatty acid in a transgenic positive plant, quickly and conveniently screening out an edited plant and an unedited plant, so that the editing efficiency and the miss effect of a specific CRISPR / Cas9 gene editing system are quickly, accurately and efficiently determined.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to CRISPR / Cas9 gene editing, in particular to a method for evaluating CRISPR / Cas9 gene editing efficiency or off-target frequency. Background technique [0002] The full name of CRISPR is "Clustered Regularly Interspaced Short PalindromicRepeats", that is, clustered, regularly spaced short palindromic repeats, which is a site in the genome that contains multiple short repeats. It acts as a kind of acquired immunity. The flanking sequence analysis of the CRISPR cluster found that there was a polymorphic family gene near it. The proteins encoded by this family all contain functional domains that can interact with nucleic acids (with activities such as nuclease, helicase, integrase, and polymerase), and work together with the CRISPR region, so they are named CRISPR-associated genes (CRISPR-associated genes). associated), abbreviated as Cas. Cas genes have co-evolved with CRISPR and togeth...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/20
Inventor 朱丽颖郑月萍徐雪珍段芊芊郑志富
Owner ZHEJIANG FORESTRY UNIVERSITY
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