Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

Abstract

Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes.

Pooled extracellular receptor-ligand interaction screening using CRISPR activation

“…CRISPRa upregulates the transcription of gRNA-specified genes [ 13 – 19 ]; however, few studies have directly investigated its effects on protein abundance within individual cells and, in the case of cell surface receptors, whether the proteins are displayed on the plasma membrane [ 20 ]. Starting with the synergistic activation mediator approach of Konermann et al [ 14 ], which uses both a VP64 transcriptional activator fused to the C-terminus of a deactivated Cas9 (dCas9) protein and recruitment of p65 and HSF1 through an MS2 fusion protein to stem loop structures added to modified gRNAs, we generated a single plasmid that incorporated all these elements (Fig.…”

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

“…Above all, continuous development toward greater precision in using drugs to control biological events is always desired for biomedical research and potential clinical applications in a safer and more effective manner. Temporal control and dose-dependent control of the CRISPR/Cas9 machinery using HIT-Cas9, together with other HIT devices we have reported recently for transcriptional programming based on CRISPR/Cas9 and those based on transcription activator-like (TAL) effectors, 31 , 32 would open broad avenues toward many applications as a comprehensive toolbox. Further optimization of the these designs and future development of additional systems using engineered SpCas9 with distinct PAM requirements 33 or Cas9 from other species 5 will further enhance their performances and expand the repertoire of genomic loci they can modify.…”

CRISPR-Mediated Approaches to Regulate YAP/TAZ Levels

“…This is accomplished by placing dCas9, fused to a transcriptional activator or inhibitor, under the control of an inducible promoter, allowing for the tunable activation of CRISPR transcriptional modulation over time. Several plasmids encoding for dCas9 fused to transcriptional activators or repressors are publically available, as are plasmids containing CRISPR transcriptional modulation elements under the inducible control of both light and chemical activation [39, 40]. …”