Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found 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 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 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 are biased toward homonucleotide insertions. Using inverted PAM 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 events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events 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 reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

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.

Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/ deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENsto DNA may interfere with binding of key components of DNA repair complexes, favoring either classical-or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w

Gene editing with engineered nucleases introduce double‐strand breaks that are repaired by error‐prone nonhomologous end‐joining (NHEJ). In article number 1900126, Sara G. Trimidal et al. propose that the length and type or resulting indels can now be controlled by editing with different engineered nucleases or by manipulating the expression of NHEJ genes.