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 mutated and unedited alleles, whereas PCR performed using lower MgCl2 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 biallelic KO mutants harbouring indels using PCR (Bindel-PCR).
Highlights
Www.nature.com/scientificreports recombination with a homology arm), non-homologous end-joining (NHEJ) [which involves no homology or only 1–3 nucleotides of homology at the junction]6, or microhomology-mediated end-joining (MMEJ) [which involves alignment of microhomologous sequences (1–4 nt) internal to the broken ends before joining]7
To design the primers used for Bindel-PCR for detecting biallelic mutants, we first scanned for the sites that had been frequently genome-edited after recognition by guide RNA (gRNA) and subsequent cleavage by Cas[9]; for this, we used data obtained from our recent study [for mouse endothelin-1 gene (Et1) (Sakurai et al, unpublished data)] and other studies [for rat tyrosinase gene (Tyr), Rosa[26] (Gt(ROSA)26S), signal regulatory protein-α gene (Sirpa), and cytochrome P450 family 2 subfamily D gene (Cyp2d)[15], and for mouse fibroblast growth factor-10 gene (Fgf10)11]
We developed a novel method, Bindel-PCR, that enables rapid identification of biallelic indel mutants among CRISPR/Cas9-mediated genome-edited animals having indels at a region within 5 bp 5′ upstream of the proto-spacer adjacent motif (PAM)
Summary
Www.nature.com/scientificreports recombination with a homology arm), non-homologous end-joining (NHEJ) [which involves no homology or only 1–3 nucleotides (nt) of homology at the junction]6, or microhomology-mediated end-joining (MMEJ) [which involves alignment of microhomologous sequences (1–4 nt) internal to the broken ends before joining]7. Genome-edited organisms and samples such as cells/embryos obtained through HDR can be readily identified because their rapid detection is possible through PCR performed using primers corresponding to the specific sequence in the inserted DNA fragment and the use of restriction enzymes that can selectively recognise the resulting PCR products[11,12]. The T7 endonuclease 1 (T7E1)-based assay and the Surveyor enzyme mismatch cleavage assay have been most frequently used to scan for indels triggered by engineered nucleases[13] These methods are based on the identification of heteroduplex DNA formed after melting and hybridizing mutant and WT alleles, and the methods exploit the use of enzymes that can cleave heteroduplex DNA at mismatches formed by single or multiple nucleotides. We show proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp[1], Ramp[3], and Rosa[26] mouse samples carrying various types of indels
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