A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Summary

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

Abstract

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

Introduction

CRISPR-Cas9 which stands for "clustered regularly interspaced short palindromic repeats-associated protein 9" was first discovered as part of studies on bacterial adaptive immunity^1,2. Today, CRISPR/Cas9 has become the most recognized tool for programmable gene editing and different iterations of the system have been developed to allow transcriptional and epigenetic modulations³. This technology enables the highly precise genetic manipulation of almost any sequence of DNA⁴.

The essential components of any CRISPR gene e....

Protocol

1.The DD-Cas9 vector

1. Obtain DD-Cas9 vector from Addgene (DD-Cas9 with filler sequence and Venus (EDCPV), Plasmid 90085).
   ​NOTE: This is a lentiviral DD-Cas9 plasmid with a U6 promoter that drives the single guide RNA (sgRNA) transcription while the EFS promoter drives the DD-Cas9 transcription. The DYKDDDDK sequence (flag-tag) is present at the C-terminal of Cas9 followed by 2A self-cleaving peptide (P2A) that separates DD-Cas9 and modified fluorescent protein Venus (mVenus).

Discussion

The CRISPR/Cas9 technology has revolutionized the capability of functionally interrogate genomes². However, the inactivation of genes often results in cell lethality, functional deficits, and developmental defects, limiting the utility of such approaches for studying gene functions⁷. Additionally, constitutive expression of Cas9 may result in toxicity and the generation of off-target effects⁶. Different approaches have been developed to temporally co.......

Materials

Name	Company	Catalog Number	Comments
100mM DTT	Thermosfisher
10X FastDigest buffer	Thermosfisher	B64
10X T4 Ligation Buffer	NEB	M0202S
colorimetric BCA kit	Pierce	23225
DMEM, high glucose, glutaMax	Thermo Fisher	10566024
FastAP	Thermosfisher	EF0654
FastDigest BsmBI	Thermosfisher	FD0454
Flag [M2] mouse mAb	Sigma	F1804-50UG
Genomic DNA extraction kit	Macherey Nagel	740952.1
lipofectamine 2000	Invitrogen	11668019
Phusion High-Fidelity DNA Polymerase	NEB	M0530S
oligonucleotides	Sigma Aldrich
pMD2.G	Addgene	12259
polybrene	Sigma Aldrich	TR-1003-G
psPAX2	Addgene	12260
QIAquick PCR & Gel Cleanup Kit	Qiagen	28506
secondary antibodies	LICOR
Shield-1	Cheminpharma
Stbl3 competent bacterial cells	Thermofisher	C737303
SURVEYOR Mutation Detection Kit	Transgenomic/IDT
T4 PNK	NEB	M0201S
Taq DNA Polymerase	NEB	M0273S
α-tubulin [DM1A] mouse mAb	Millipore	CP06-100UG