Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering

Abstract

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance

“…Bacteria and archaea acquire short pieces, or spacers, from these invading nucleic acids and incorporate them within their genomes, where they serve as molecular memory (Bolotin et al, 2005). During subsequent infections, these short pieces are transcribed as part of the CRISPR array; after transcription and maturation, CRISPR RNA (crRNA) can help guide the Cas9 endonuclease to scan the invading DNA and cleave the target sequence (Nunez et al, 2016). The Cas9 endonuclease cleaves the target sequence at a site preceding the protospacer-associated motif (PAM), which is NGG for Streptomyces pyogenes Cas9 (Wright et al, 2016) ( Figure 2C ).…”

Naturally Occurring Off-Switches for CRISPR-Cas9

“…Based on these and other considerations, the performance and safety of CRISPR-Cas9 applications could be greatly improved if Cas9 activity could be more effectively controlled. Several groups have devised methods to activate CRISPR-Cas9 genome editing in response to specific cues, including light-inducible and drug-inducible Cas9 activity (Nihongaki et al, 2015; Nunez et al, 2016; Wright et al, 2015). However, a robust, specific, and genetically-encodable “off-switch” for Cas9 activity has not yet been identified.…”

Evolution of a split RNA polymerase as a versatile biosensor platform

“…The development of programmable DNA binding domains such as TALEs (Transcription activator-like effectors) and dCas9 have revolutionized 1- and 2-hybrid approaches 8,13–15 . However, a challenge with n-hybrids is that each system needs to be carefully tuned and optimized for each new interaction, and more complex multicomponent systems, such as 3-hybrids, often lack in sensitivity and signal-to-noise 16,17 .…”