Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.

ABSTRACT: We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl₂ concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl₂ concentrations (1.5-2.5 mM) should generate PCR products derived from both mutate

SUBMITTER: Sakurai T 

PROVIDER: S-EPMC6617447 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature