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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0818-7) contains supplementary material, which is available to authorized users.

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems

“…This technique can identify a set of putative PAMs, although it remains limited by the availability of matching phage or plasmid DNA sequences in genomic databases, and obtained hits may include mutated escape-PAMs. More recent efforts have developed high-throughput, experimental screens to determine functional PAMs based on the depletion of a target plasmid or on the introduction of a double-stranded break in vitro (Jiang et al, 2013a; Karvelis et al, 2015; Pattanayak et al, 2013). However, plasmid removal screens are based on an irreversible binary event, implicitly measure the frequency of escape from killing, and require high library coverage to quantitatively identify depleted PAM sequences.…”

“…However, plasmid removal screens are based on an irreversible binary event, implicitly measure the frequency of escape from killing, and require high library coverage to quantitatively identify depleted PAM sequences. Separately, in vitro DNA cleavage screens require the purification of active protein-RNA complexes and can be highly sensitive to the assay conditions (Karvelis et al, 2015). Furthermore, in vitro screens that rely on adaptor ligation are incompatible with Type I systems that cleave and degrade DNA (Pattanayak et al, 2013; Zetsche et al, 2015).…”

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

“…First, Cpf1 is guided by a crRNA, whereas Cas9 uses dual-guide RNAs, a crRNA and a trans -activating crRNA (Deltcheva et al, 2011). Second, Cpf1 recognizes a T-rich PAM (Zetsche et al, 2015), whereas Cas9 favors a G-rich PAM (Jinek et al, 2012; Fonfara et al, 2014; Karvelis et al, 2015; Ran et al, 2015). Third, Cpf1 generates staggered ends at its PAM-distal region (Zetsche et al, 2015), whereas Cas9 creates blunt ends at its PAM-proximal region (Garneau et al, 2010).…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…Cpf1-mediated cleavage is guided by a single and short (42-44 nt) crR-NA [19], in contrast to Cas9 that uses both crRNA and tracrRNA [20]. Cpf1 recognizes a T-rich PAM at the 5′-end of the protospacer sequence [19], in contrast to 3′-G-rich PAM recognition by Cas9 [21,22]. More importantly, Cpf1 makes a staggered double-strand break resulting in five-nucleotide 5′-overhangs distal to the PAM site [19], whereas Cas9 creates blunt ends proximal to the PAM site [8].…”