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A method for detecting the activity of a double-strand break generating reagent

A technology of double-strand breaks and reagents, applied in biochemical equipment and methods, measuring devices, biomaterial analysis, etc., can solve the problem of limiting the application of gene editing methods, difficult to distinguish PCR amplification products from original templates, and increasing the risk of gene editing, etc. question

Active Publication Date: 2022-04-26
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, engineered nucleases, including CRISPR / Cas, all have off-target activities, that is, they cause cleavage at other genomic locations that have certain sequence differences from the target site, and sometimes even cause chromatin rearrangement, which greatly increases gene The risks brought about by editing greatly limit the application of gene editing methods in clinical treatment
Some methods to detect the editing efficiency and specificity of nucleases have been developed in the prior art, for example, targeted high-throughput sequencing is widely used to assess indels in genomic fragments amplified by PCR (Mali et al., 2013 ), these data can be used to roughly estimate nuclease cleavage efficiency, but these methods do not estimate nuclease specificity; whereas LAM-HTGTS identifies off-target sites by using genome-wide translocations of target loci as baits ( Frock et al., 2015) have the ability to detect its editing specificity
However, this method first performs 80 cycles of linear amplification to generate multiple copies of the original DNA fragment, which will make it difficult to distinguish the PCR amplification product from the original template
In addition, restriction enzyme blockade during library preparation leads to underestimation of uncut or fully repaired target fragments and small inserts, thereby failing to quantify DSB repair products around the target site (Hu et al., 2016), so this method cannot be used efficiently. Quantitative analysis of cutting efficiency

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  • A method for detecting the activity of a double-strand break generating reagent
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  • A method for detecting the activity of a double-strand break generating reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0078] Materials and methods

[0079] PEM-seq procedure and data analysis

[0080] 1. Primer extension

[0081] All biotin-labeled primers (see Table 2 for sequences) (Sangon, Shanghai) were set within 100 bp from the cleavage site. Repeated annealing and denaturation of the primers and 20 μg of sonicated genomic DNA (0.3-2 kb), the specific conditions are as follows: 95°C for 3 minutes; 95°C for 2 minutes, annealing at the annealing temperature Ta for 3 minutes, 5 cycles ( See Table 1 for details); annealed at Ta for 3 minutes. Bst polymerase 3.0 (NEB) was then added for primer extension: 10 minutes at 65°C, 5 minutes at 80°C. After that, 1.2×AxyPrep Mag PCR Clean Up bead (Axygen, USA) was added to remove excess biotinylated primers. The purified product was heated to 95°C for 5 minutes, then rapidly cooled on ice for 5 minutes to denature the DNA. Finally using Dynabeads TM MyOne TM Sterptavidin C1 (Thermo Fisher) enriches for biotin-labeled extension products.

...

Embodiment 2

[0134] Example 2 PEM-seq can detect off-target hotspots of CRISPR / Cas9 very sensitively

[0135] This example compares with the LAM-HTGTS method, which identified 33 off-target sites in HEK293T cells using SpCas9 targeting the RAG1A site (Frock et al., 2015). Each PEM-seq library was constructed using about 20 μg of CRISPR / Cas9-treated genomic DNA, and the extension primers were designed to be within 200 bp of the target cleavage site. Experiments were repeated three times for hotspot analysis of translocation junctions ( figure 1 B and 1C); hotspots with a high sequence similarity to the target site and / or defined PAMs occurring in at least two replicates were considered off-target sites (Frock et al., 2015).

[0136] A total of 53 off-target sites were identified by PEM-seq, including 24 new sites not identified by LAM-HTGTS, while 4 weak sites identified by LAM-HTGTS were lost ( figure 1 B-D and Table S2). In order to verify the authenticity of these off-target sites, 8 ...

Embodiment 3

[0138] Example 3 PEM-seq can quantitatively analyze the editing ability of CRISPR / Cas9

[0139] This example tests the ability of PEM-seq to analyze all gene editing events, especially the quantitative analysis. The events generated after gene editing can be divided into: chromosomal translocation, insertion and deletion (indel) and germline (germline). Chromatin translocation is the joining of two independent double-strand breaks (DSBs), such as those that occur between a target DSB and a DSB cleaved off-target by Cas9, or with a basal level of DSB naturally present in the genome; indels are detected by Cas9 The cleaved ends are reconnected through the in vivo DNA repair mechanism, but due to incomplete repair, the repaired sequence is different from the original sequence, resulting in insertion or deletion of the sequence ( figure 2 A). The so-called "germline" is that the target fragment is not cut or completely repaired after cutting. In the case of complete repair, the...

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Abstract

The invention relates to a method for detecting the activity of a double-strand break generating reagent, in which the dual detection of the editing efficiency and specificity of the reagent is realized through the design of an extension primer and a molecular beacon of a random sequence.

Description

technical field [0001] The invention provides a method for simultaneously detecting the editing efficiency and specificity of double-strand break generating reagents, especially engineered nucleases. Background technique [0002] In genetic engineering, appropriate genome editing tools are often required to operate on target genes or genome fragments, and the ability to cause double-strand breaks in target genome sequences is the basic function of various genome editing tool projects. One of the widely used double-strand break reagents is engineered nuclease, which includes meganucelases, zinc finger nucleases, transcription activator-like effector nucleases (Transcription activator-like effector nucleases, TALENs) and CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins), etc., can precisely achieve targeted editing at almost any genomic location, thereby realizing precise modification of the genome. Among them, CRISPR / Cas, deriv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/44C12N9/22C12N15/63
CPCC12Q1/6869C12Q1/44C12N9/22C12N15/63G01N2333/922C12Q2535/122C12Q2521/327C12Q1/68C12Q1/66C12N15/85
Inventor 胡家志尹健行刘孟竺刘阳
Owner PEKING UNIV
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