A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR‐Cas9

Abstract

Several genome engineering applications of CRISPR‐Cas9, an RNA‐guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small‐molecule systems lack wide dynamic range, have background activity in the absence of the small‐molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high‐intensity light. We previously reported a small‐molecule‐controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next‐generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low‐intensity light while rendering the small‐molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.

Genome-editing prodrug: Targeted delivery and conditional stabilization of CRISPR-Cas9 for precision therapy of inflammatory disease

“…Thus, upon the successful intracellular delivery of NanoProCas9, the nuclease only becomes functional when dsCas9 is stabilized by TMP. Previous reports have proved that the proteasomal degradation and nuclease activity of dsCas9 act in a concentration-dependent manner, where sufficient TMP concentration is required to recover the full nuclease activity of dsCas9 ( 11 ). Furthermore, given the fact that the restriction of the nuclease activity to a narrow temporal window is highly desirable to minimize the off-target events, we believe that the lower off-target activity contributed by NanoProCas9, as opposed to wild-type Cas9, is mainly due to the exhaustion of TMP released from NanoProCas9 that further results in the confined time window of genome-editing activity.…”

“…In general, chemical control of Cas9 functions takes advantages of the ability of tailored small molecules to activate or inhibit Cas9 nuclease activity. Several chemical control strategies, such as destabilized Cas9 (dsCas9) systems ( 11 , 12 ), self-splicing intein-Cas9 systems ( 13 ), and dimerization of split-Cas9 systems ( 14 ), have been designed in an attempt to increase spatiotemporal specificity of CRISPR-Cas9 genome editing. As opposed to light, small molecules can be ideally delivered in an organ-specific manner to reach the deep tissues from the site of administration, eliminating the critical issue of penetration depth that optical regulation suffers ( 15 ).…”

Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo

“…The DHFR DD system is also reversible in that TMP can be washed out in vitro and metabolized or excreted in vivo . This generalized model system has been confirmed to be effective in controlling the abundance of numerous fusion proteins13, 14, 15, 16, 17, 18, 19 in several tissues, including the brain10, 20, 21 and the eye,11, 22 in a spatial, temporal, and dose-dependent manner.…”

Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing

“…Fusing protein domains can also be used to control the expression of the Cas protein, which can limit off-target activity by spatially and/or temporally limiting where and/or when the CRISPR-Cas system is active. To this end, efforts have focused on the fine control of CRISPR-Cas systems by inserting or fusing active domains to Cas proteins, including inducible Cas proteins (or split Cas proteins) by intervening protein (intein) insertion [38,39], photoregulation of Cas proteins [40,41], and light-induced or chemically induced CRISPR-Cas systems [42,43]. Furthermore, these systems can be combined for synergetic applications.…”