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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2018-02, Vol.115 (9), p.E2040-E2047
• Main Authors: Lemos, Brenda R., Kaplan, Adam C., Bae, Ji Eun, Ferrazzoli, Alexander E., Kuo, James, Anand, Ranjith P., Waterman, David P., Haber, James E.
• Format: Article
• Language: English
• Subjects: Binding; Biological Sciences; Cells; Chromosomes - ultrastructure; Cleavage; Clonal deletion; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; Dimerization; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA End-Joining Repair; DNA Ligase ATP - metabolism; DNA polymerase; DNA Repair; DNA-Binding Proteins - metabolism; DNA-directed DNA polymerase; Double-strand break repair; Endonuclease; Endonucleases - metabolism; Gene Deletion; Gene sequencing; gRNA; HO endonuclease; INDEL Mutation; Insertion; Inspection; Ku Autoantigen; Mammalian cells; MRE11 protein; Non-homologous end joining; Nuclease; Nucleotide sequence; Plasmids - metabolism; PNAS Plus; Promoter Regions, Genetic; Repair; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; Saccharomycetales - genetics; Sequence Analysis, DNA; Studies; Technology; Yeast
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1716855115

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.