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Biotechniques. 2017 Jun 1;62(6):268-274. doi: 10.2144/000114556.

Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines.

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Advanced Analytical Technologies, Inc., Ankeny, IA.


The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events. The only current method to determine the zygosity of an edited gene in a diploid organism is DNA sequencing, which is costly and time-consuming. Here, we describe the development of a T7 endonuclease I (T7EI)-based heteroduplex cleavage assay, along with statistical models relating the percentage of cleaved DNA to the zygosity of a mutation, that provides a rapid screening step prior to DNA sequencing. By isolating candidates likely to contain the desired zygosity for the edited gene, our screening method can decrease the number of clones requiring DNA sequencing.


CRISPR; T7 endonuclease I; gene editing; heteroduplex cleavage assay; mutation detection

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