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

Duy P Nguyen,Jonathan S Weissman,Bruce R Conklin,Luke A Gilbert,Steven J Mayerl,Brian H Lee,James A Wells,Yuichiro Miyaoka

doi:10.1038/ncomms12009

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.

Highlights

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
Redesigning the single-guide RNA (sgRNA) to retarget virtually any DNA sequence leads to the ease of construction of RGN reagents for genome editing purposes
Efforts to interrogate cellular dynamics of gene transcription, epigenetic modifications and genome organization would be greatly aided by techniques that are capable of dynamically controlling Cas9/dCas[9] variants without background activity

Summary

Introduction

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 Cas[9] protein from bacterial type II CRISPR-Cas systems has recently been adapted to induce sequence-specific alterations in many genomic contexts including mammalian genomes[3,4,5,6] In this Cas9-based RNA-guided nuclease (RGN) system, DNA recognition requires a single-guide RNA (sgRNA) that contains the first 20 nucleotides complementary to the target sequence and a protospacer adjacent motif sequence immediately upstream of the target sequence[3,5,6,7,8,9]. With the recent advent of dCas9-based techniques in transcriptional regulation and epigenetic modifications, we anticipate a need for a robust and rapidly inducible system for simultaneous orthogonal activation of different CRISPR systems to provide dynamic control of distinct loci. Efforts to interrogate cellular dynamics of gene transcription, epigenetic modifications and genome organization would be greatly aided by techniques that are capable of dynamically controlling Cas9/dCas[9] variants without background activity

Methods

Results

Conclusion