Figure 4. S. rosetta preferentially introduces genome-edited mutations from DNA templates.

(A) Schematic of a gRNA targeting SpCas9 to a genomic locus of interest. A gRNA (cyan, knobs indicate 5’ ends) that encodes a 20 nt targeting sequence from the sense strand of a genomic locus (black) hybridizes with the antisense strand (dark gray). SpCas9 (light gray) introduces a double-stranded break at the genomic locus (carets), 3 bp upstream of a protospacer adjacent motif (PAM, orange). (B) We designed a panel of repair oligonucleotides to test the preferred substrates for repairing double-stranded breaks introduced by SpCas9 at rosetteles exon 4. Oligonucleotide repair templates containing the PTS sequence (green) were delivered as single-stranded DNA in the sense (S) or anti-sense (A) orientations and as a double-stranded template (D) to test which most efficiently templated DNA repair at the SpCas9 cleavage site. (C) SpCas9 stimulated the introduction of PTS mutations from DNA templates. Repair templates with a PTS (from panel B) were delivered in the presence and absence of SpCas9 (+/–). A ~ 450 bp fragment surrounding the rtls^PTS1 cleavage site was amplified from cells that had been transfected the previous day to prepare deep sequencing libraries for quantifying the frequency of PTS insertions (green) or insertions/deletions from error prone editing (black). Each experiment was performed three independent times (dots; mean and standard deviations are shown with lines). The dotted line indicates the limit of detection of the sequencing, based on a 6-base, randomized barcode. Upon transfection with the SpCas9 RNP, 10x more mutations from repair templates (1–2%, green dots) were detected than untemplated insertions or deletions (black dots). In the absence of SpCas9, mutations generated from a double-stranded template, but not single-stranded templates, were rarely (<0.1%) and unreliably (2 of 3 trials) found.

Figure 4—figure supplement 1. Characterization of editing outcomes at the rosetteless locus with different types of repair templates.

(A) Double-stranded DNA repair templates (black indicates homology arms from the sense strand, gray indicates homology arms from the antisense strand and green is the PTS as in Figure 4) were designed with phosphorylated 5’ ends (indicated with open circles at the 5’ end and a ‘p’ in template names; closed circles indicate unphosphorylated ends) or three phosphorothioate bonds between bases at the 5’ end (indicated with asterisks in diagrams and a ‘t’ in template names). We hypothesized that phosphorylated templates would be more susceptible to nucleases and phosphorothioate bonds less susceptible (Renaud et al., 2016; Yu et al., 2020), altering their utility as repair substrates in vivo, yet S. rosetta used all double stranded templates with similar efficiency (see panel C). (B) We also designed a panel of single-stranded repair templates (colors as in panel A) that lacked 5’ or 3’ arms (Paix et al., 2017) and found that both arms of homology are required for efficient template-mediated genome editing in S. rosetta (see panel C). (C) A comparison of DNA repair templates revealed that S. rosetta efficiently uses double stranded DNA templates during DNA repair and requires both arms of homology for single-stranded DNA templates. Frequencies of alleles containing either the PTS (green) or insertion/deletion mutations (black) are shown for genome editing experiments based on each of the templates described in panels A and B. Genome editing in the presence of double-stranded DNA templates favored template-directed DNA repair. The use of phosphorylated double-stranded DNA templates or double-stranded DNA templates with phosphorothioate bonds (see panel A) did not increase editing efficiency over unmodified double-stranded DNA templates. We also found that removing 5’ or 3’ homology arms from single stranded templates (see panel B) almost completely eliminated efficient editing as compared to single-stranded templates with both homology arms (Figure 4C). Each editing experiment was performed three independent times. (D–E) An aggregate analysis of insertion/deletion mutations identified in deep sequencing of genome editing experiments. (D) A histogram shows the length and frequency of insertion and deletion mutations. (E) A sequence alignment or representative insertion and deletion mutations from each size of insertion/deletion mutations. Notably, the most frequent deletions (8–10 bases) occur at dinucleotide repeats, suggesting that microhomologies may promote deletions after double-stranded breaks. (F) An extreme example of templated repair suggests that S. rosetta may incorporate larger insertions. One mutation identified in deep sequencing shows an 88-base insertion, with the insertion featuring two PTS sequences with an intervening region that has some homology to sequences to the left (thick line) and right (dotted line) of the double stranded break. Although we are unsure of the mechanism that led to this mutation, its presence suggests that large mutations could be incorporated into S. rosetta via genome editing.