Rapidly Prototyping CRISPR Biology and Technologies with an All-E. coli Txtl System

CRISPR-Cas systems represent widespread adaptive immune systems in bacteria and archaea that have become versatile technologies driving applications in gene therapy, diagnostics, antimicrobials, functional genetic screens, and more. The vast diversity of these immune systems and their broad applicability as technologies have spurred efforts into their discovery, characterization, and implementation. However, traditional cell-based and in vitro techniques represent a notable bottleneck. Here, I will describe how cell-free transcription-translation (TXTL) systems can be employed to greatly accelerate the prototyping of CRISPR biology and technologies. I will show how an all-E. coli TXTL system can be used to rapidly and scalably characterize target recognition by CRISPR nucleases, elucidate rules for guide RNA design, and identify different inhibitors of nuclease activity. I will also illustrate the power of TXTL using two examples from our group. In one example, we developed RNA-responsive guide RNA switches that only drive nuclease activity in the presence of a selected RNA sequence. We used TXTL to devise general design rules for these switches, allowing us to apply these rules to generate functional switches that respond to synthetic and native RNAs in E. coli. In another example, we developed two means to rapidly reverse gene repression by catalytically-dead CRISPR nucleases through anti-CRISPR proteins or upstream-binding guide RNAs. TXTL allowed us to elucidate design rules for either means and translate insights into orthogonal repression and de-repression circuits as well as dynamic feedback controllers. Overall, these examples highlight an important and growing use of the ease, speed, and scalability of TXTL.