CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Abstract

Harnessing CRISPR-Cas9 technology has provided an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We have analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in the budding yeast Saccharomyces cerevisiae and find that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a one base pair (+1) insertion that appears to result from Cas9 creating a 1-bp 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which were biased toward homonucleotide insertions. Using inverted PAM (iPAM) sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9::gRNA complex influences subsequent NHEJ processing. As with well-studied events induced by the site-specific HO endonuclease, CRISPR-Cas9 mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events, however, are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells (van Overbeek et al., 2016) reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

“…18 out of 95 screened Myr2 sgRNA‐treated clones (18%) and 14 out of 181 screened PPYP6 sgRNA‐treated clones (8%) contained group 1 ERV mutations at the RNA level (Tables S3 and S4). Among the Myr2 sgRNA‐treated and mutated clones, the majority possessed an identical 1 bp insertion upstream of the ATG start codon (Tables S4 and S5), which likely resulted from staggered CRISPR‐Cas9 cleavage (Lemos et al, ). No clone treated with the PPYP6 sgRNA acquired a mutation disrupting the PPYP motif.…”

A new inducible CRISPR‐Cas9 system useful for genome editing and study of double‐strand break repair in Candida glabrata

“…Furthermore, the protocol allows for better control of the cutting step, so that it can also be used, for example, to examine repair of DSBs in the same fashion that it is done in S . cerevisiae (Lemos et al, ). In this work, we have used the system to confirm that faithful NHEJ is a major repair mechanism of chromosomal DSBs in C .…”

“…In yeasts, the system has been used, first and foremost, in S . cerevisiae , for genome engineering (DiCarlo et al, ) but also for the study of the repair of DSBs (Lemos et al, ) and for mating‐type switching (Xie et al, ). Several versions of the CRISPR‐Cas9 systems have also been designed for C .…”

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

“…The remaining events (80.4%) corresponded to extensive rearrangements involving retrotransposon LTRs. However, given that there is no transposon or transposon remnant in the close proximity of the LYS2 locus, the authors could not retrieve LTR rearrangements (Lemos et al, 2018). This strongly suggests that rearrangements observed heavily depend on the surrounding chromosomal location where the DSB is made.…”