Principle of Bindel-PCR for identifying CRISPR/Cas9-induced biallelic mutants. (a) Outline of Bindel-PCR. 1st PCR is carried out under standard concentrations of Mg2+ (1.5–2.5 mM) for confirming successful amplification of a target region. For the 2nd PCR, the sense primer was set corresponding to the sequence that is recognised by gRNA and cleaved by Cas9 immediately upstream of the PAM of the target gene, whereas the reverse primer was set on a downstream sequence distant from the PAM. PCR performed using this primer set and the 1st PCR products as template under standard conditions should generate PCR products (~200 bp) derived from both indel-mutated alleles and unedited alleles. Contrastingly, when the concentration of MgCl2 used for PCR is lowered, annealing between a primer and the mutated target sequence becomes unstable, and under this condition, PCR products derived from unedited alleles, such as WT allele, should be generated preferentially. Mg2+ is recognised as not only a cofactor for DNA polymerase activity, but also a promoter of complex formation between primers and DNA template26. Blue boxes, sequences recognised by forward and reverse primers used for 1st PCR; green box, sequence recognised by crRNA; red box, PAM sequence. (b) Flowchart of detection of biallelic KO mutants by using Bindel-PCR. (c) The evaluation rule for indels appearing in the target sequence is shown using mouse Et1 gRNA-recognizing nt region as an example. Green nt correspond to the sequence recognised by gRNA-Et1. Red nt correspond to the PAM sequence. nt −1 to –23 correspond to the 5′ upstream region of the PAM. nt +1 to +15 correspond to the 3′ downstream region of the PAM. Arrows indicate the site of indels classified. WT, wild-type. (d) Analyses of sites frequently showing CRISPR/Cas9-induced indels. Data shown are from our previous study (Sakurai et al., unpublished data) on mouse Et1 and from other studies [Yoshimi et al. (2016) for rat Tyr, Rosa26, Sirpa, and Cyp2d and Hashimoto and Takemoto (2015) for mouse Fgf10]. *Includes the samples with indels at nt (−1) as well as those with indels including a part of the PAM. Et1, endothelin-1; Tyr, tyrosinase; Rosa26, Gt(ROSA)26S; Sirpa, signal regulatory protein-α; Cyp2d, cytochrome P450, family 2, subfamily d; Fgf10, fibroblast growth factor-10.