Figure 3—figure supplement 3.

Figure 3—figure supplement 3.

(A) Comparison between results obtained with a proofreading (Phusion Hot Start II) or a non-proofreading (REDTaq) DNA polymerase for three target loci (A, B, C) of slc24a5 amplified with the PCR primers used for sequencing (std, standard) or when one is replaced by a headloop primer (HL). Samples were uninjected controls. Orange arrowheads mark the 300 bp ladder band. (B) Headloop primer designs, using slc24a5 locus G as an example. To perform headloop PCR, the forward or reverse primer from a previously verified primer pair is modified with a 5’ tag sequence and used in conjunction with its unmodified partner. The sequence of the headloop tag is selected so that the predicted Cas9 cleavage site (dashed line) is located towards the 5’-end of the tag. (left) If the modified primer and the gRNA binding site are in the same direction (headloop tag is added to the forward primer and gRNA binding site is on the 5’–3’ genomic strand), the reverse-complement of the gRNA binding site is sufficient (grey underlay). (right) If the modified primer and the gRNA binding site are in opposite directions (headloop tag is added to the reverse primer while gRNA binding site is on the 5’–3’ genomic strand), a sequence that includes the protospacer adjacent motif (PAM, orange font) and shifted from the gRNA binding site is sufficient. In both cases, after second strand elongation, the tag is able to bind the target sequence and direct elongation (hatched sequences) to form a hairpin, suppressing exponential amplification of the wild-type haplotype. Framed: headloop tag; grey font: gRNA binding site; grey underlay: headloop tag binding site.