Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Homologous recombination (HR) repairs DNA double-strand breaks and maintains genome stability. HR between linked, direct repeats can occur by gene conversion without an associated crossover that maintains the gross repeat structure. Alternatively, direct repeat HR can occur by gene conversion with a crossover, or by single-strand annealing (SSA), both of which delete one repeat and the sequences between the repeats. Prior studies of different repeat structures in yeast and mammalian cells revealed disparate conversion:deletion ratios. Here, we show that a key factor controlling this ratio is the distance between the repeats, with conversion frequency increasing linearly with the distances from 850 to 3800 bp. Deletions are thought to arise primarily by SSA, which involves extensive end-processing to reveal complementary single-strands in each repeat. The results can be explained by a model in which strand-invasion leading to gene conversion competes more effectively with SSA as more extensive end-processing is required for SSA. We hypothesized that a transcription unit between repeats would inhibit end-processing and SSA, thereby increasing the fraction of conversions. However, conversion frequencies were identical for direct repeats separated by 3800 bp of transcriptionally silent or active DNA, indicating that end-processing and SSA are not affected by transcription.

Gene conversion is a common outcome of double-strand break (DSB) repair in yeast. Prior studies revealed that DSB-induced gene conversion tracts are often short (<53 bp), unidirectional, and biased toward promoter-proximal (5') markers. In those studies, broken ends had short, non-homologous termini. For the present study we created plasmid x chromosome, chromosomal direct repeat and allelic recombination substrates in which donor alleles carried mutant HO sites (HOinc--not cleaved) at the same position as cleavable HO sites in recipient alleles. In these substrates, broken ends are almost completely homologous to donor alleles, differing only at the three HOinc mutations. These mutations serve as markers very close to, or within, the four-base overhang produced by HO nuclease. We identified extremely short tracts (<12 bp) and many tracts were highly directional, extending <2 bp on one side of the DSB. We thought that terminal homology would promote bidirectional tracts, but found instead that unidirectional tracts were more frequent. Interestingly, substrates with terminal homology displayed enhanced 3' conversion, and in several cases conversion bias was reversed toward 3' markers. These results are discussed in relation to factors that may influence tract length and directionality, including heteroduplex DNA formation, transcription, replication and mismatch repair.

Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nucleaseinduced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.DNA double-strand breaks (DSBs) are potentially lethal events that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). If left unrepaired, DSBs can lead to chromosome loss or cell death. DSBs are caused by ionizing radiation, X-rays, free radicals, chemicals, and nucleases and may occur at stalled replication forks (30). Although DSB repair can be accurate, misrepair can have serious consequences. Genomic rearrangements arising during DSB repair can lead to loss of heterozygosity and, ultimately, carcinogenesis through the activation of proto-oncogenes or inactivation of tumor suppressor genes (4, 45). HR appears to be essential for maintaining genome stability. Cells with defects in HR proteins such as BRCA1/2, XRCC2/3, and other RAD51 paralogs exhibit high levels of genomic instability (6,18,28,42,43,52,68). DSBs are a critical type of DNA damage; unlike single-strand breaks, which have a readily available template for repair, DSB repair by HR requires a search for a homologous template.Repetitive elements make up one-third of the mammalian genome and consist of coding DNA, and noncoding DNA such as satellites that can exist in thousands of copies. Repetitive sequences are scattered throughout the genome, including SINE and LINE elements, and ribosomal RNA gene repeats. HR between linked or unlinked repetitive elements can result in a variety of rearrangements including translocations, duplications, and deletions (45).When DNA is broken, there are many potential homologous sequences that could be used as a repair template, including linked or unlinked repeats, sister...

CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements.PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.