Harnessing bacterial immune system for targeted genome engineering

Bacteriophages infect bacteria in order to reproduce and often pose a lethal threat to bacteria. To cope with onslaughts of phages bacteria evolved multiple defense barriers that interfere with nearly every step of virus life cycle. Taken together, these defense barriers constitute a primitive immune system that protects bacteria against invading viruses. In prokaryotes restriction-modification system acts as an innate immune system that uses methyl-tag to discriminate between self and non-self DNA, while CRISPR-Cas system functions as an adaptive arm that memorizes the invader by inserting fragments of the foreign DNA into the host chromosome. Class 2 CRISPR-Cas systems based on the single effector protein, as exemplified by Cas9, rely on the protospacer adjacent motif (PAM) sequence to discriminate between host and alien DNA. Deciphering the molecular mechanisms underlying the CRISPR-Cas immunity paved the way for development of novel tools for targeted genome engineering. Class 2 effector nucleases that provide CRISPR-Cas immunity are abundant in prokaryotes. To explore this largely uncharacterized diversity of the Class2 nucleases for genome editing applications, we established a phylogeny-guided bioinformatic approach and developed biochemical screens for the rapid identification and characterization of the PAM and guide RNA requirements of new nucleases. This approach permitted the rapid characterization of many Cas9 orthologs with diverse PAM sequence requirements. Next, using the same biochemical screen we detected PAM and gRNA requirements for several miniature CRISPR-associated proteins. Taken together, our results demonstrate that Class 2 effector nucleases provide a rich source of biochemical and biophysical diversity that may be beneficial for genome editing.