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Scientists Develop Assay for Characterizing Editing-Related Features of Novel CRISPR/Cas9 Systems

NEW YORK (GenomeWeb) – Scientists from Lithuania's Vilnius University and DuPont Pioneer have described a method to characterize essential features of CRISPR/Cas9 systems in bacteria that could open up new genomic areas for editing.

Led by Virginijus Siksnys of Vilnius University and Mark Cigan of DuPont Pioneer, the scientists described an in vitro method to find the protospacer adjacent motifs (PAMs) of new and existing CRISPR/Cas9 systems. PAMs are short sequences of DNA that must immediately flank the target area of the genome being edited. Without knowing a Cas9's preferred PAM sequence, researchers can't predict which genomic sequences the protein can target.

Using plasmid libraries containing randomized PAMs, the scientists were able to reproduce the canonical PAM preference for the gold standard Cas9 of Streptococcus pyogenes as well as two S. thermophilus CRISPR proteins. They also published newly characterized PAM preferences for a Cas9 protein from Brevibacillus laterosporus that edits genomes in vitro and in plants. The study ran yesterday in Genome Biology.

Though there are more than 1,000 Cas9 sequences available in genomic databases, "methods to ascertain the PAM sequence requirement for new Cas9 proteins are limited," the authors wrote. PAMs are typically found by analyzing the CRISPR sequences in the bacterial genome that match bacteriophage genomes, since CRISPR is a bacterial defense system to protect against phage attack; however, only a few of these sequences match known phage sequences, limiting the utility of the different Cas9 variants for genome editing applications.

To find the PAM for a given Cas9 variant, the scientists designed plasmids with a randomized PAM adjacent to the guide RNA target sequence. They used two libraries, one with five randomized base pairs in the PAM (yielding 1,024 possible combinations) and one with seven base pairs (16,384 possibilities).  The Cas9 and guide RNA were introduced in vitro. Cleaved plasmids were captured by ligating adapters to the free ends, amplified by PCR with a primer targeting the adapter, and sequenced to reveal the PAM preference of the Cas9 being investigated.

The assay correctly reported the recognized PAM sequence NGG for the Cas9 from S. pyogenes. It also enabled the scientists to edit DNA with the Cas9 from B. laterosporus, a previously uncharacterized CRISPR/Cas9 system, in maize cells.

Characterizing these Cas9 properties essential to genome editing applications could open the door to a new generation of CRISPR systems and enable orthologous, multiplex genome editing, according to the researchers.