In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Summary

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Abstract

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Introduction

One of the challenges for cancer research in the post-genomic era is to mine the vast amount of genome data for causal gene mutations and to identify nodes in the gene network that can be targeted therapeutically. While bioinformatic analyses have helped immensely towards these goals, establishing efficient in vitro and in vivo models is a prerequisite to decipher the complexity of biological systems and disease states and for enabling drug development. While conventional transgenic mouse models have been used extensively for in vivo cancer genetics studies, their cost-, time- and labor-intensive nature has largely prohibited the systematic analysis of the hundreds of....

Protocol

This protocol was approved and performed in accordance with IACUC of University of Toronto.

1. Design and cloning of pooled CRISPR libraries

1. Select 4-5 sgRNAs targeting mouse genes of interest from resource such as the Broad Institute sgRNA designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) or CHOPCHOP server (https://chopchop.cbu.uib.no). Select an equal number of non-targeting sgRNAs from e.g., Sanjana et al.⁹ to generate an equally sized non-targeting controls gRNA library.
2. While constructing the sgRNA libraries, make sure there is enough coverage for each sg....

Results

Figure 1A shows the design of the oligonucleotides for multiplexing several custom CRISPR libraries in a cost-effective manner in a single 12k or 92k oligo chip. Once the sgRNAs (blue color coded) are selected, the oligonucleotides are designed with restriction sites (orange colored BsmBI) and library specific PCR primer pairs (green color coded). Several libraries can be designed by using unique combination of primer pairs for multiplexing in a single oligo chip. When PCR amplifying the lib.......

Discussion

CRISPR/Cas9 genome editing has been widely used in in vitro and in vivo studies to investigate gene functions and cellular processes. Most in vivo studies utilize CRISPR/Cas9 gene edited cells grafted into an animal model (allograft or xenograft). While this is a powerful tool to study cancer genetics and cellular functions, it still lacks the native tissue microenvironment and might elicit wounding and/or immune responses.

To overcome these challenges, several groups have pioneered direct in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 micron filter	Sigma	S2HVU02RE
12k or 92k oligo chip	Customarray Inc. (Genscript)
15 cm cell culture plates	Corning
293FT	Invitrogen	R70007
293NT	Systems Biosciences	LV900A-1
Alkaline phosphatase	NEB	M0290L
Amplicillin	Fisher Scientific	BP1760-25
ATP	NEB	9804S
BsmBI	NEB	R0580L
Chromic gut suture	Covidien
Deep sequencing (Next-Seq or Hi-Seq)	Illumina
DNA-cleanup kit	Zymo Research	D4008
DNAesy Blood and Tissue DNA extraction kit	Qiagen	69506
Endura electrocompetent cells	Lucigen	60242-1
Glass Capillaries	Drummond	3-000-203-G/X
HEK293T cells	ATCC	CRL-3216
High-Speed Centrifuge	Beckman Coulter	MLS-50
LB Agar	Wisent Technologies	800-011-LG
Micropipette puller	Sutter Instrument	P97
Mineral oil	Sigma	M5904
Mini-prep plasmid Kit	Frogga Bio	PDH300
Mouse oxygen anaesthesia system	Visual Sonics
Nanoject II micromanipulator	Drummond
NEBuffer 3.1 (Buffer for BsmBI)	NEB	R0580L
Needle sharpener	Sutter Instrument	BV-10
Oligo cleanup kit	Zymo research	D4060
PAGE purified illumina sequencing primer	IDT DNA
PEI (polyethyleneimine)	Sigma	408727-100ML
Permoplast modeling clay
Petridish with central opening	Visual Sonics
pMD2.G	Addgene	12259
psPAX2	Addgene	12260
Q5 Polymerase 2x Master mix	NEB	M0494L
Qubit Fluorometric Quantification	Invitrogen	Q33327
Semicircular Silicone plug	Corning
Silicone membrane	Visual Sonics
T4 DNA ligase	NEB	M0202L
Ultra-centrifuge tubes	Beckman Coulter	344058
Vevo2000 ultrasound system	Visual Sonics