用于检测 CRISPR 诱导的 Indels 的 PCR 诱导突变-限制性片段长度多态性方法。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY
CRISPR Journal Pub Date : 2023-12-01 Epub Date: 2023-12-05 DOI:10.1089/crispr.2023.0047
Lydia Angelopoulou, Electra Stylianopoulou, Konstantinos Tegopoulos, Ioanna Farmakioti, Maria Grigoriou, George Skavdis
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引用次数: 0

摘要

由于基于 CRISPR 的技术被广泛用于敲除细胞系和生物体中的基因,因此需要开发可靠、经济、快速的方法来鉴定完全突变的克隆。在此背景下,我们提出了一种名为 "PCR诱导突变-限制性片段长度多态性(PIM-RFLP)"的新策略。PIM-RFLP 可以评估编辑细胞池的编辑效率,并有效鉴定完全突变的单细胞克隆。它的基础是通过诱变 PCR 在野生型等位基因的 PCR 产物中创建一个限制性酶变性裂解位点,然后按照标准 RFLP 程序将其与含 indel 的等位基因区分开来。PIM-RFLP 非常容易获得,可在一天内完成,而且在检测完全突变的克隆方面似乎优于 Sanger 测序解旋算法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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