A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing.

  title={A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing.},
  author={Kaiwen Ivy Liu and Muhammad Nadzim Bin Ramli and Cheok Wei Ariel Woo and Yuanming Wang and Tian Yun Zhao and Xiujun Zhang and Guo Rong Daniel Yim and Bao Yi Chong and Ali Gowher and Mervyn Zi Hao Chua and Jonathan Jung and Jia Hui Jane Lee and Meng How Tan},
  journal={Nature chemical biology},
  volume={12 11},
CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low… 

Rapid Control of Genome Editing in Human Cells by Chemical-Inducible CRISPR-Cas Systems.

The use of an inducible CRISPR-Cas9 system, termed iCas, is described, which is developed to enable rapid and tight control of genome editing in mammalian cells.

Modulating Cas9 activity for precision gene editing.

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

Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers.

A simple tunable CRISPR controller is reported in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim, and significantly reduced off-target activity in mammalian cells is shown.

Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9

RNA linkers are developed to combine theophylline- and 3-methylxanthine-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli and reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.

Protein Engineering Strategies to Expand CRISPR-Cas9 Applications

Recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.

Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening

The optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control and represents a significant upgrade on existing functional genomics toolbox is reported.

Precision Control of CRISPR-Cas9 Using Small Molecules and Light.

A perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas 9 is provided.

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

HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes and exerted selective, titratable, rapid and reversible response to drug induction.



Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

It is shown here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site- specific double-strand DNA breaks using timed delivery ofCas9-guide RNA ribonucleoprotein (RNP) complexes.

Rationally engineered Cas9 nucleases with improved specificity

Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, it is demonstrated that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off- target effects and maintain robust on-target cleavage, which could be broadly useful for genome-editing applications requiring a high level of specificity.

Genetic Screens in Human Cells Using the CRISPR-Cas9 System

A pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library is described and it is shown that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs.

Inducible in vivo genome editing with CRISPR/Cas9

It is shown that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively.

High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

It is found that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface, and off-target cleavage of CRISPR-associated (Cas)9-based RGNs is characterized.

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

This system is engineer to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA).

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.

Delivery of purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells is delivered and RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off- target mutations associated with plasmid transfection at off-target sites.

Multiplex Genome Engineering Using CRISPR/Cas Systems

Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.