Figure 5.

Comparison of Cas9 cleavage site positions for N-terminal HBTH tagging of mRNA RNMT. A, schematic layout of the RNMT ORF region surrounding the start codon. Two gRNAs were selected using gRNA Designer (28). gRNA1 directs Cas9 cleavage 3′ of the start codon, and microhomology regions can be located next to the cut site to direct insertion. gRNA2 guides Cas9 cleavage to the 5′ UTR. Therefore, microhomology sequences need to be chosen so that part of the 5′ UTR is removed during the repair process and the tag is fused seamlessly to the RNMT1 ORF. B, HBTH-RNMT was detected using RGS6H and RNMT antibodies. gRNA2, which mediates intra-ORF cleavage, yielded more homozygous knockins.