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J Biotechnol. 2015 Nov 20;214:128-32. doi: 10.1016/j.jbiotec.2015.09.029. Epub 2015 Sep 28.

Research of methods to detect genomic mutations induced by CRISPR/Cas systems.

Author information

1
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: zyppwk@vip.qq.com.
2
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: 739757163@qq.com.
3
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: 392943348@qq.com.
4
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: 1012687173@qq.com.
5
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: wruan@henu.edu.cn.
6
School of Life Science, Henan University, Kaifeng 475004, PR China. Electronic address: huangtian2007@126.com.
7
School of Life Science, Henan University, Kaifeng 475004, PR China; Institute of Bioengineering, Henan University, Kaifeng 475004, PR China. Electronic address: gscao@henu.edu.cn.

Abstract

The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90 min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.

KEYWORDS:

Amplified fragment length polymorphism; CRISPR/Cas system; Genome editing; Quantitative PCR high-resolution melting; SURVEYOR assay

PMID:
26419205
DOI:
10.1016/j.jbiotec.2015.09.029
[Indexed for MEDLINE]

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