A practical guide for characterization of novel CRISPR-Cas systems with Pro-CRISPR factorsOA

The emergence of advanced genome editing technologies has revolutionized research in life sciences,offering an unprecedented way to uncover unknown biological functions and innovative therapeutic strategies.Among all genome editing tools,CRISPR-Cas-based technologies play a pivotal role in this revolution,particularly Class 2 effectors such as Cas9 and Cas12,owing to their high efficacy and ease of programmability.With the advancements in genome sequencing and metagenomics,an increasing number of novel CRISPR-Cas systems have been discovered,including those found in extreme environments and viruses.Furthermore,recent studies have revealed an unexpected role of non-Cas accessory genes,such as the Tn7-like transposon and Pro-CRISPR factors(Pcr),in conferring additional functionalities to the CRISPR system,providing new insights into the understanding of CRISPR-mediated bacterial immunity and advancing the development of genome editing technologies.Therefore,it is essential to develop comprehensive methods for characterizing the Cas proteins and Pro-CRISPR factors with a growing diversity.In this protocol,we provide a method encompassing protein purification,biochemical characterization,validation of protein-protein interactions,and preliminary in vivo functional assays in bacteria for Cas nuclease and its associated Pro-CRISPR factor.We hope this protocol will not only assist in the characterization of the CRISPR-Cas system,but also provide valuable guidance for the characterization of other nucleases or nucleic acid modification systems.