Directed Evolution of CRISPR Endoribonucleases Using Phage-Assisted Continuous Evolution (PACE)

A panel of sequence-specific RNA-cleaving enzymes would enhance our ability to manipulate RNA structure and function and aid in the analysis of coding and non-coding RNAs found in human and other genomes. We have developed a selection system that enables the phage-assisted continuous evolution (PACE) of RNA-cleaving enzymes. Using PACE, we have evolved variants of the CRISPR endoribonuclease Csy4 that demonstrate altered substrate specificity.