(734b) A Synthetic-Biology Approach to Rapidly Characterize Novel Crispr-Cas Systems

CRISPR-Cas systems have rapidly transitioned from curious defense systems to powerful and versatile genetic tools. A central driver has been the ease in designing synthetic CRISPR RNAs that guide the system’s Cas proteins to bind and cleave target DNA. The only design rules are ensuring the spacer portion of the CRISPR RNA is complementary to a target sequence and selecting target sequences flanked by a protospacer-adjacent motif (PAM). Once directed to the target sequence, the system can be utilized for a range of applications that include genome editing, gene regulation, sequence-specific antimicrobials, and many others. To date, these applications have overwhelming relied on the CRISPR-Cas system from the bacterial pathogen Streptococcus pyogenes. However, thousands of other CRISPR-Cas systems have been found in nature and offer distinct attributes and capabilities. The challenge is rapidly characterizing these systems in order to generate the next generation of CRISPR technologies.

We developed a simple genetic circuit in Escherichia coli to rapidly identify the PAM, an essential design feature that varies widely between systems and has proven difficult to predict a priori. The designed circuit relies on CRISPR-Cas systems modified for gene repression. As part of the circuit, CRISPR-based repression is converted into the upregulated expression of a fluorescent reporter through an inverter gate. By introducing libraries of random sequences in the location of the PAM, functional PAM sequences can be rapidly identified using fluorescence-activated cell sorting followed by next-generation sequencing. We applied this screen to a collection of Type I systems and Type II systems—the two types that require PAMs and target DNA. The screen reproduced known PAMs for well-characterized systems and revealed PAMs for never-before characterized systems. The screen also revealed flexibility in the spacing between the target sequence and PAM, suggesting that CRISPR-Cas systems have a greater propensity for off-target effects than previously realized. Overall, our synthetic circuit offers a simple and rapid means of characterizing novel CRISPR-Cas systems, with a step toward bringing the remarkable diversity of these natural defense systems to the forefront of biotechnology.