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A genome editing detection method, kit and application

A genome editing and detection method technology, applied in the field of gene editing and detection, can solve the problems of cumbersome experimental steps and affecting accuracy, and achieve the effect of simple process and reliable quantitative results

Active Publication Date: 2021-02-02
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors believe that the above experimental steps are cumbersome, and they use the PCR amplification product of the genomic target DNA region instead of the genomic DNA itself to quantify the editing efficiency
It is well known that sequence and length-dependent biases introduced during PCR amplification will inevitably affect detection accuracy

Method used

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  • A genome editing detection method, kit and application
  • A genome editing detection method, kit and application
  • A genome editing detection method, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170] The design of value guard base in the embodiment 1 getPCR

[0171] In order to better apply the getPCR technology, in this embodiment, the design rules of the guarded bases are studied. Since most indels appear near the nuclease cleavage site, and indels smaller than 15 bp account for the main part, in addition, in order to better distinguish the indel sequence from the wild-type sequence, the number of bases in this embodiment is relatively small Insertions or deletions were investigated. In view of this, the inventors designed and constructed 26 plasmids, each with an indel mutant of 1-15bp, to simulate the nuclease-induced genome editing of the HOXB13 gene in vivo ( figure 2 a).

[0172] In this embodiment, two series of guarded bases are designed, and they respectively have one to eight guarded bases ( Figure 7 a-c), from which representatives with ideal amplification efficiency were screened out ( figure 2 b) to further check their discriminative ability, th...

Embodiment 2

[0175] Embodiment 2 runs the parameters of getPCR

[0176] Another factor that needs to be determined is the optimal parameter for getPCR operation. In this embodiment, the annealing temperature during the getPCR reaction is studied. For the four groups of guarded bases designed in Example 1, as the annealing temperature increases, compared with the indel template containing mismatched bases, the ability of getPCR to specifically amplify the wild-type template DNA increases significantly ( image 3 a-d). However, when the annealing temperature was increased above 4 °C above the Tm value, the PCR efficiency began to drop significantly. Since PCR amplification usually prefers the best PCR efficiency, this embodiment systematically evaluates the selectivity of each guarded base at the best PCR efficiency ( image 3 e-h). Interestingly, no matter how many guard bases or total bases the primer has, the best selectivity is usually observed when the annealing temperature is about ...

Embodiment 3

[0179] Example 3 Research on the Accuracy of GetPCR Quantitative Genome Editing

[0180] figure 2 Shown in a are the plasmids used to model insertion-deletion mutations (indels) caused by genome editing, which were first used to assess the ability of getPCR to quantify genome editing efficiency. In this example, twenty-six indel plasmids were mixed in equal parts, and then mixed with wild-type plasmids in a specific ratio to simulate indel frequencies of 0%, 20%, 40%, 60%, 80% and 100%. The indel frequency of the mixture was quantified and compared by getPCR and the classic Surveyor method. When the indel frequency is not higher than 20%, the quantitative results of the Surveyor method can truly reflect the expected value. However, as the indel frequency increases further, the observed value gradually deviates from the expected value ( Figure 4 a-b). In contrast, all 12 getPCR strategies using different guard bases, whether carrying 3, 4 or 5 guard bases on the guard bas...

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Abstract

The disclosure belongs to the field of genome editing efficiency detection, and specifically relates to a genome editing detection method, kit and application. In view of the defects of gene editing efficiency detection methods in the prior art, such as Sanger, NGS, and methods based on mismatch-specific nucleases, etc. have defects such as complicated operation, high cost, and insufficient detection accuracy, the present disclosure provides a new method , called getPCR, based on Taq DNA polymerase specificity and real-time PCR technology, quantifies the wild-type DNA in the genome to be tested, and confirms the genome editing efficiency by calculating the percentage of wild-type DNA. This disclosed research provides the design rules of the corresponding guarded bases and optimized getPCR operation parameters. It has been verified that the method has good detection accuracy and is easy to operate. It can be applied to all genome editing methods to quantify genome editing efficiency, and can also be applied to Screening of single cell clones.

Description

technical field [0001] The disclosure belongs to the field of gene editing detection, and specifically relates to a method for indirectly confirming the probability of genome editing by amplifying and quantifying the proportion of wild-group DNA in a genome, and its application in genome editing efficiency evaluation and monoclonal screening. Background technique [0002] The information disclosed in this Background section is only intended to increase the understanding of the general background of the disclosure, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes prior art that is already known to those skilled in the art. [0003] CRISPR / cas9 is a currently mainstream genome editing technology, and its gene modification effect is related to guide RNA (sgRNA). In the CRISPR / cas9 system, the Cas9 nuclease is guided to the target DNA containing the protospacer adjacent motif (PAM) by sgRNA, and then cuts the two ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6858
CPCC12Q1/6851C12Q1/6858C12Q2521/101C12Q2531/113C12Q2545/113C12Q2525/131C12Q2545/114
Inventor 黄启来李博
Owner SHANDONG UNIV
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