A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Abstract

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.

Fig. 1. Cas9 diversity and characterization approach.

a Phylogenetic representation of the diversity provided by Cas9 orthologs. Type II-A, B, and C systems are color-coded, red, blue, and green, respectively. Distinct phylogenetic clades are numbered I–X. Those selected for the study are indicated with a black dot. Cas9s whose structure has been determined are also designated. b Biochemical approach used to directly capture target cleavage and assess protospacer adjacent motif (PAM) recognition. Experiments were assembled using Cas9 protein produced by IVT.

Fig. 2. Cas9 tracrRNA sequence and secondary structure similarity.

Circles are scaled based on the number of sequences belonging to each covariance model (CM) and colored according to the designated cluster. The width of the connecting lines indicates the percentage of similarity or relatedness among CMs. Representative tracrRNAs from each cluster are indicated with the associated color. CMs not assigned to a cluster are in gray.

Fig. 3. Cas9 protospacer adjacent motif (PAM) interacting (PI) domain similarity.

Cas9 PI domains clustered by their pairwise sequence similarity. Sequences were clustered using CLANS (BLAST option). Lines connect sequences with P value ≤ 1e − 11. Line shading corresponds to P values according to the scale in the top-right corner (light and long lines connect distantly related sequences). For details on how P values are calculated, please see the “Methods” section. Major clusters are shown in bold. Cluster 1 was so named to emphasize that it contains the first experimentally characterized Cas9, Spy. Clusters 2–10 were named beginning from the one with the most members. Different clusters are indicated, and PAM sequences recognized by members of each cluster are highlighted with the associated color. The Cas9 which belongs to the same clade is outlined by a black dashed line. Sequences having known structures are marked red; their PDB code is shown in parentheses.

Fig. 4. Activity of Cas9 orthologs at varying temperatures.

The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled double-stranded DNA (dsDNA) substrates. Cleaved fragments were quantitated and are represented in a heatmap a showing overall activity at temperatures ranging from 10 °C to 68 °C. The intensity of the blue color indicates the proportion of substrate cleaved. Source data are provided in the Source Data file. b Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with >50% activity at 53 °C is summarized in a heatmap and plotted as the proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Points represent the mean  ± SEM of at least three independent experiments. Green shading highlights the temperature range above 53 °C. c Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with <25% activity at 25 °C is summarized in a heatmap and plotted as a proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Red shading highlights the temperature range below 25 °C Points represent the mean ± SEM of at least three independent experiments.

Fig. 5. Target DNA cleavage patterns produced by Cas9 orthologs.

Cleavage sites and resultant double-stranded DNA (dsDNA) ends are depicted as heatmaps that show the proportion of cleaved ends recovered by DNA sequencing at each position of a target DNA. The intensity of the blue color indicates the proportion of mapped cleavage ends. a Control digests using restriction enzymes showed that blunt ends, 5′-overhangs and 3′-overhangs might be recovered with our approach. TS indicates the top strand; BS indicates the bottom strand. b Spy Cas9 and Sau Cas9 cleaved DNA ends. Heatmaps represent mapped cleavage ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and protospacer adjacent motif (PAM) sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. c Blunt and staggered-end cleavage. Examples of blunt, one base 5′-overhang staggered cleavage, and multiple base 5′-overhang cleavage are depicted as heatmaps that show the proportion of cleaved ends as the averages at each position in five different dsDNA targets. The position of the DNA bases and PAM sequences is depicted above the heatmaps. NTS indicates a non-target strand; TS indicates the target strand. Source data are provided in Supplementary Data 5.