Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

Abstract

Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.

Multidimensional chemical control of CRISPR–Cas9

“…Finely tuned control of Cas9 levels is important 8 , as high Cas9 leads to elevated off-target genomic alterations. Rapid disabling of Cas9 activity after a desired genomic modification is also valuable to prevent off-target activity 9 .…”

Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

“…Our approach was to employ a self-destructive kamikaze CRISPR/Cas system that disrupts the CRISPR/Cas enzyme itself after the active protein has been expressed. Unlike other approaches, most of which act to control the activity of the CRISPR/Cas system via chemical, 23,24 and biophysical 25,26 modulation of Cas9, our kamikaze CRISPR/Cas system can significantly reduce accumulation of Cas9, and thus off-target cleavage, without dramatically compromising the efficiency of on-target editing. This approach is similar to that used by Merienne and colleagues, 27 who demonstrated that progressively inactivating the nuclease using a Cas9 self-inactivating editing system resulted in a lower frequency of off-target cleavage in human iPSCs-derived neurons in vitro and in mouse brains in vivo .…”

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

“…Such strategies promise to mitigate offtarget effects and allow the study of complex biological perturbations that require temporal or spatial resolution [9]. To date, most of the progress in this area has been focused on switching the activity of the Cas9 protein using chemical [10][11][12][13][14][15][16] or optical [17][18][19] inputs. A general issue with these approaches is that all target genes are regulated in the same manner, although this limitation can be addressed with orthogonal CRISPR-Cas9 systems [16,20,21].…”