Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

Abstract

To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.

Fig. 1

Schematic for identification of PAM preferences by Cas9 cleavage in vitro. a Initial plasmid library with randomized PAM (green box) is cleaved with Cas9 complex and 3′ dA overhangs are added. b Adapters with 3′ dT overhang (blue box) are ligated to both ends of the cleavage product. c Primers are utilized to enrich for PAM-sided cleaved products by PCR. d After PCR enrichment, DNA fragments are purified and Illumina compatible anchors and barcodes are ‘tailed-on’ through two rounds of PCR (gray boxes) and Illumina deep sequenced

Fig. 2

PAM preferences for S. pyogenes (a), S. thermophilus CRISPR3 (b), and S. thermophilus CRISPR1 (c) Cas9 proteins. Frequency of nucleotides at each PAM position was independently calculated using a position frequency matrix (PFM) [21] and plotted as a WebLogo [23]

Fig. 3

Identification of Type II CRISPR-Cas elements in Brevibacillus laterosporus SSP360D4 CRISPR-Cas system. a An illustration of the genomic DNA region from the Type II CRISPR-Cas system from Brevibacillus laterosporus SSP360D4. b Comparison of Type II CRISPR array repeat sequences identified in Brevibacillus laterosporus SSP360D4. c The ‘direct’ and ‘reverse’ tracrRNA and CRISPR array transcriptional scenarios for the Type II CRISPR-Cas system from Brevibacillus laterosporus SSP360D4. d An agarose gel with reaction products, indicating that only the ‘direct’ sgRNA (dir sgRNA), but not the ‘reverse’ sgRNA (rev sgRNA) support plasmid library cleavage in combination with the Cas9 endonuclease originating from Brevibacillus laterosporus SSP360D4

Fig. 4

PAM preferences and cleavage positions of Brevibacillus laterosporus SSP360D4 (Blat) Cas9 enzyme. Blat Cas9 PAM preferences when 1 μg of library DNA was cleaved with 0.5 nM or 50 nM Cas9-sgRNA complex (a), extended out to position 10 by shifting the protospacer target by 3 bp (b). Frequency of nucleotides at each PAM position was independently calculated using a position frequency matrix (PFM) [21] and plotted as a WebLogo [23]. c Cleavage rates of supercoiled plasmid DNA substrates containing mutations (shown in red) in GTCCCGAA PAM sequence. All data points are mean values from ≥3 independent experiments. Error bars are given as S.D. d Run-off sequencing from both sense and anti-sense directions of plasmid DNA cleaved with Blat Cas9

Fig. 5

Brevibacillus laterosporus Cas9 promotes NHEJ mutations in maize. a Top 10 most prevalent types of NHEJ mutations detected with Blat Cas9 in exon 4 of the Ms45 gene. A black arrow indicates the expected site of cleavage; mutations are highlighted in red; lower case font indicates an insertion; ‘-’ indicates a deletion. b Comparison of Spy and Blat Cas9 NHEJ mutation frequencies at three protospacer identical target sites in maize. NHEJ mutations were detected by deep sequencing 3 days after transformation. Error bars represent SEM, n = 3 particle gun transformations. Cas9 only is the negative control and represents the average (across all three target sites) background frequency of mutations resulting from PCR amplification and sequencing