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

• Published in: Scientific reports Vol. 9; no. 1; pp. 9923 - 14
• Main Authors: Sakurai, Takayuki, Kamiyoshi, Akiko, Takei, Norio, Watanabe, Satoshi, Sato, Masahiro, Shindo, Takayuki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.07.2019; Nature Publishing Group
• Subjects: 101/47; 38/70; 38/77; 631/1647/1511; 631/1647/277; 64/60; 82/29; Alleles; Animals; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA-directed DNA polymerase; Embryos; Female; Gene Editing; Genomes; High-throughput screening; Humanities and Social Sciences; Magnesium chloride; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; multidisciplinary; Mutants; Mutation; Nucleotide sequence; Nucleotides; Polymerase chain reaction; Polymerase Chain Reaction - methods; Receptor activity modifying proteins; Receptor Activity-Modifying Protein 1 - antagonists & inhibitors; Receptor Activity-Modifying Protein 1 - genetics; Receptor Activity-Modifying Protein 3 - antagonists & inhibitors; Receptor Activity-Modifying Protein 3 - genetics; Ribonucleic acid; RNA; RNA, Untranslated - antagonists & inhibitors; RNA, Untranslated - genetics; Science; Science (multidisciplinary); Spacer; Taq Polymerase - genetics; Taq Polymerase - metabolism; Thermus - enzymology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-46357-8

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 MgCl 2 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 MgCl 2 concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl 2 concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3 , and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of b iallelic KO mutants harbouring indel s using PCR (Bindel-PCR).