Figure 4. csRNP cutting proximal to insertion site is more efficient and allows knock-in at non-expressed genes.

(A) Schematic showing the relative position of guide RNAs in the 3’ UTR. The first set of gRNA (gRNA1) cuts proximal to the stop codon, the second set (gRNA 2) cuts distal. Average distance of cut site from the insertion site is shown for both sets of gRNAs against 50 TFs. (B) Representative V5 ICC images of Sox3, Sox5, and Sox8 V5 knock-in bulk populations obtained with gRNA 1 (left panels) and gRNA 2 (right panels) are shown; % V5 knock-in efficiency for each experiment is shown at the right bottom of V5 panels (numbers in white). (C) Heatmap representation of V5 knock-in efficiency obtained with gRNA1 and gRNA2 for different genes, color code is shown on the right. (D) Example of sequencing traces using the bulk populations of V5-tagged cells for Sox8 and Foxj3. Alignment with the expected TF-V5 chimeric sequence is shown.

Figure 4—figure supplement 1. V5 tag-specific PCR amplification of bulk populations gene-edited using first set of gRNAs.

(A) Schematics showing location of gRNAs, first set of gRNAs cut proximal to the stop codon and shown in red. (B) PCR strategy is shown, a V5 tag-specific reverse primer and a TF exon-specific forward primer were used to amplify HDR-edited regions for each of the 50 TFs. (C) Agarose gel analysis of PCR product. Gene names are indicated at the top of each lane, lanes marked with ‘+’ denote successful knock-in of the V5-encoding sequence at the target gene (based on the expected size of PCR products), 1kb + DNA ladder is marked ‘L’.

Figure 4—figure supplement 2. V5 tag-specific PCR amplification of bulk populations gene-edited using second set of gRNAs.

(A) Schematics showing location of gRNAs, second set of gRNAs cut distal to the stop codon and shown in red. (B) PCR strategy is shown, a V5 tag-specific reverse primer and a TF exon-specific forward primer were used to amplify HDR-edited regions for each of the 50 TFs. (C) Agarose gel analysis of PCR product. Gene names are indicated at the top of each lane, lanes marked with ‘+’ denote successful knock-in of the V5-encoding sequence at the target gene (based on the expected size of PCR products), 1kb + DNA ladder is marked ‘L’.

Figure 4—figure supplement 3. Sanger sequencing confirms error-free insertion of V5-encoding sequence at the C-terminus of expressed and non-expressed genes.

Schematics at the top shows the PCR amplification of HDR-edited loci using a TF exon-specific forward primer and a V5 tag-specific reverse primer. Sequencing traces from the respective PCR amplicons were aligned with the expected TF-V5 chimeric sequence.