A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.

CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.

Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

Temporal and rheostatic control of genome editing with a chemically-inducible Cas9.

Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo.

Multi-input chemical control of protein dimerization for programming graded cellular responses.

Chemical and optogenetic methods for post-translationally controlling protein function have enabled modulation and engineering of cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed, we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs using a suite of engineered reader proteins to provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program a variety of cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our versatile tool set will enable researchers to post-translationally program mammalian cellular processes and to engineer cell therapies.

Rheostatic Control of Cas9-Mediated DNA Double Strand Break (DSB) Generation and Genome Editing.

We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic time-course experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.