Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition

• Published in: PloS one Vol. 13; no. 2; p. e0192181
• Main Authors: Anderson, Emily M, McClelland, Shawn, Maksimova, Elena, Strezoska, Žaklina, Basila, Megan, Briner, Alexandra E, Barrangou, Rodolphe, Smith, Anja van Brabant
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02-02-2018; Public Library of Science (PLoS)
• Subjects: Bacteria; Biology and Life Sciences; Biotechnology; Cell cycle; CRISPR; Deoxyribonucleic acid; DNA; Engineering and Technology; Eukaryotes; Flexibility; Genes; Genetic modification; Genome editing; Genomes; Genomics; Lactobacillus gasseri; Mammalian cells; Multiplexing; Nuclease; Nucleases; Nutrition; Physiological aspects; Plasmids; Proteins; Research and Analysis Methods; RNA; United States; North Carolina; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0192181

While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.