A Cas9 with PAM recognition for adenine dinucleotides

Abstract

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5[Formula: see text]-NAAN-3[Formula: see text] PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.

Fig. 1. Identification of features from natural PAM divergence through bioinformatics.

a Sequence alignment of SpyCas9, its QQR variant, and SmacCas9. The step in the underlining red line marks the joining of SpyCas9 and SmacCas9 to construct a SpyMac hybrid. The sequence logo (Weblogo online tool) immediately below the alignment depicts the conservation at 11 positions around the PAM-contacting arginines of SpyCas9. b The domain organization of SpyCas9 juxtaposed over a color-coded structure of RNA-guided, target-bound SpyCas9 (PDB ID 5F9R). The two DNA strands are black with the exception of a magenta segment corresponding to the PAM. A blue–green–red color map is used for labeling the Cas9 PI domain and guide spacer sequence to highlight structures that confer sequence specificity and the prevalence of intra-domain contacts within the PI. c A sequence logo generated online (WebLogo) that was input with putative PAM sequences found in Streptococcus phage and associated with close SmacCas9 homologs.

Fig. 2. Validation of SmacCas9 recognition for adenine dinucleotide PAM sequences.

a Chromatograms representing the PAM-SCANR based enrichment of variant-recognizing PAM sequences from a 5${}^{\prime }$-NNNNNNNN-3${}^{\prime }$ library. b SYBR-stained agarose gels showing in vitro digestion of 10 nM 5${}^{\prime }$-NAAN-3${}^{\prime }$ substrates upon 16 minutes of incubation with 100 nM of purified ribonucleoprotein enzyme assemblies. Arrows distinguish banding of the cleaved products from uncleaved substrate (top band). Matrix plots summarize cleaved fraction calculations, which were carried out in a custom script for processing gel images. Samples were performed in independent biological duplicates (n = 2). c Time course measurements of target DNA substrate cleavage for SmacCas9 and SpyMac. d DNA substrate cleavage plotted as a function of 0.25:1, 1:1, and 4:1 molar ratios of ribonucleoprotein to target for wild-type SpyCas9 and hybrid SpyMac. Source data are provided as a Source Data file.

Fig. 3. Genome editing capabilities of engineered SmacCas9 variants.

a CRISPResso2 indel analysis following NGS of amplified genomic regions to assess on-target editing of iSpyMac in comparison to SpyMac and SpyCas9, on the indicated 5${}^{\prime }$-NAA-3${}^{\prime }$ and 5${}^{\prime }$-NGG-3${}^{\prime }$ PAM sequences. Samples were performed in two independent transfection replicates (n = 2). b Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies, as assessed by the TIDE algorithm, are exhibited for each labeled single or double mismatch in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. Samples were performed in two independent transfection replicates (n = 2). c CRISPResso2 genomic base editing analysis following NGS of genomic amplicons to assess conversion of cytosines to thymines by iSpyMac-BE3. Samples were performed in two independent transfection replicates (n = 2). All source data are provided as a Source Data file.