Optimized protocols for the characterization of Cas12a activities.

The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.

The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies.

Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.