Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Summary

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Abstract

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

Introduction

CRISPR-Cas, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated nuclease, consists of a single nuclease protein (e.g., Cas9) in complex with a synthetic guide RNA (sgRNA). This ribonucleoprotein complex targets the Cas9 enzyme to induce double-stranded DNA breaks at a specific genomic locus¹. Double-stranded breaks can be repaired via homology directed repair (HDR) or, more commonly, through non-homologous end joining (NHEJ), an error prone repair mechanism that results in insertion and/or deletions (INDELS) that frequently disrupt gene function¹. The efficiency and simplicity of CRI....

Protocol

The experiments outlined below should follow the institute’s Environmental Health and Safety Office guidelines.

1. Pooled CRISPR sgRNA lentiviral library plasmid amplification

1. Dilute the ready-made CRISPR sgRNA plasmid DNA library to 50 ng/μL in TE (e.g., TKOv3).
2. Electroporate the library using electrocompetent cells. Set up a total of four electroporation reactions as described below.
   1. Add 2 μL of 50 ng/μL TKO library to 25 μL of thawed electrocompetent cells to pre-chilled cuvettes (1.0 mm) on ice.
   2. Electroporate using optimal settings suggested by the manufact....

Results

Overview of genome-scale CRISPR screening workflow

Figure 1 illustrates an overview of the pooled CRISPR screening work flow, starting with infection of target cells with CRISPR library lentivirus at a low MOI to ensure single integration events and adequate library representation (typically 200- to 1000-fold). Following infection, cells are treated with the antibiotic puromycin to .......

Discussion

Due to its simplicity of use and high pliability, CRISPR technology has been widely adopted as the tool of choice for precise genome editing. Pooled CRISPR screening provides a method to interrogate thousands of genetic perturbations in a single experiment. In pooled screens, sgRNA libraries serve as molecular barcodes, as each sequence is unique and is mapped to the targeted gene. By isolating the genomic DNA from the cell population, genes causing the phenotype of interest can be determined by quantifying sgRNA abundan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 micron filter
30°C plate incubator
37°C shaking incubator
37°C, 5% CO2 incubator
5 M NaCl	Promega	V4221
50X TAE buffer	BioShop	TAE222.4
6 N Hydrochloric acid solution	BioShop	HCL666.500
95% Ethanol
Alamar blue	ThermoFisher Scientific	DAL1025
Blue-light transilluminator	ThermoFisher Scientific	G6600
Bovine Serum Albumin,Heat Shock Isolation, Fraction V. Min. 98%, Biotechnology grade	Bioshop	ALB001.250
Dulbecco's Modification of Eagles Medium	Life Technologies	11995-065	Cel culture media
Electroporation cuvettes	BTX	45-0134
Electroporator	BTX	45-0651
Endura electrocompetent cells	Lucigen	90293
Fetal Bovine Serum	GIBCO	12483-020
HEK293T packaging cells	ATCC	CRL-3216	recommend passage number <15
Hexadimethrine Bromide (Polybrene)	Sigma	H9268	Cationic polymer to enhance transduction efficiency
Hexadimethrine Bromide (Polybrene)
LB agar plates with carbenicillin
LB medium with carbenicillin
Low molecular weight DNA ladder	New England Biolabs	N3233S
Nanodrop spectrophotometer	ThermoFisher Scientific	ND-ONE-W
NEBNext Ultra II Q5 Master Mix	New England Biolabs	M0544L
Opti-MEM	Life Technologies	31985-070	Reduced serum media
Plasmid maxi purification kit	Qiagen	12963
pMD2.G (envelope plasmid)	Addgene	Plasmid #12259	lentiviral system
psPAX2 (packaging plasmid)	Addgene	Plasmid #12260	lentiviral system
Puromycin	Wisent	400-160-UG
QIAquick gel extraction kit	Qiagen	28704
Qubit dsDNA BR assay	ThermoFisher Scientific	Q32853
Qubit fluorometer	ThermoFisher Scientific	Q33226
RNAse A	Invitrogen	12091021
S.O.C recovery medium	Invitrogen	15544034
SYRB Safe DNA gel stain	ThermoFisher Scientific	S33102
Toronto KnockOut CRIPSR library (TKOv3) - Cas9 included	Addgene	Addgene ID #90203	Genome-wide CRISPR library , includes Cas9, 71,090 sgRNA
Toronto KnockOut CRIPSR library (TKOv3) - non-cas9	Addgene	Addgene ID #125517	Genome-wide CRISPR library, non-Cas9, 71,090 sgRNA
Tris-EDTA (TE) solution, pH8.0
UltraPure agarose	ThermoFisher Scientific	16500500
Wizard genomic DNA purification kit	Promega	A1120
X-tremeGENE 9 DNA transfection reagent	Roche	06 365 809 001	Lipid based transfection reagent