HIT-Cas9: A CRISPR/Cas9 Genome-Editing Device under Tight and Effective Drug Control

Abstract

The CRISPR/Cas9 enabled efficient gene editing in an easy and programmable manner. Controlling its activity in greater precision is desired for biomedical research and potential therapeutic translation. Here, we engrafted the CRISPR/Cas9 system with a mutated human estrogen receptor (ERT2), which renders it 4-hydroxytamoxifen (4-OHT) inducible for the access of genome, and a nuclear export signal (NES), which lowers the background activity. Tight and efficient drug-inducible genome editing was achieved across several human cell types, including embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), upon vigorous optimization. Optimized terminal device, which we named hybrid drug inducible CRISPR/Cas9 technology (HIT-Cas9), delivered advantageous performances over several existing designs. Such architecture was also successfully applied to an orthogonal Cas9. The HIT-Cas9 system developed in this study will find broad utility in controlled editing of potentially any genomic loci.

Genetic screens in isogenic mammalian cell lines without single cell cloning

“…Inducible CRISPR systems could be helpful, but all of them require the use of additional components, such as recombinases, degrons, dimerization domains, transcriptional activators, or transcriptional repressors, as well as small-molecule inducers, many of which have biological effects. Further, recent comparisons have shown that current systems often have substantially less activity than constitutive versions, or demonstrate leakiness; additionally, performance is typically cell-type dependent 23,24 . Thus, there is a need for a simple method to generate cells poised for gene editing, expand them with no selective pressure, and trigger efficient knockout only when ready to begin a genetic screen.…”

Drug Inducible CRISPR/Cas Systems

“…As previously discussed, Duy Nguyen et al generated a rapamycin inducible split Cas9 system for genome editing [43]. To damp its background activity, they fused ER T2 to both the splitted Cas9-FRB/FKBP partners and found that this design confer a tighter drug control of both genome editing and transactivation, consistent with findings from HIT and iCas systems [[70], [71], [72]]. However, it required two small molecules rapamycin and 4-OHT to reach the highest efficiency.…”

“…Our laboratory has developed an ER T2 based genome editing tool named Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT)-Cas9, in which 2 NES and 2 ER T2 domains are fused sequentially at the C terminal of Cas9 to deliver efficient activity upon 4-OHT induction without introducing significant background in its absence (Fig. 2C) [70]. In iCas design, 4 ER T2 domains are fused with Cas9, 2 to each terminus [71].…”

“…In iCas design, 4 ER T2 domains are fused with Cas9, 2 to each terminus [71]. In side by side experiments [70], HIT-Cas9 showed high efficiency, low background, and selective response to exogenous 4-OHT when compared with iCas [71], 4-OHT inducible-intein [64] and split-Cas9 [42]. In addition, our laboratory also developed HIT systems for inducible transcription activation by grafting ER T2 to previously existing dCas9 based transactivation devices, including direction fusion, SAM and SunTag [72].…”

“…Key properties of each system were summarized in Table 1. Analyzing these systems altogether provides interesting observations: First, it appears that subjecting CRISPR/Cas9 to drug control commonly enables greater precision and less off-target activity, regardless of its working mechanism [70]. This promises an important advantage of such approaches for scenarios, such as somatic gene therapy, where precision and safety are essential.…”