Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Summary

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

Abstract

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Introduction

Stable genetic alterations provide an advantage over transient methods of cellular perturbation, which can be variable in their efficiency and duration. Genomic editing has become increasingly common in recent years due to the development of target specific nucleases, such as zinc-finger nucleases^1,2,3,4,5, transcription activator-like effector nucleases (TALENs)^6,7,8,9 and RNA-gu....

Protocol

1. Identification of Homology Regions Around the Desired Deletion

NOTE: Only necessary if using selection-based editing.

1. Select two regions, initially 1.5-2 kb, on either side of the desired deletion locus, which will serve as homology arms in the HDR template (Figure 1A). Identify regions that lack BsaI recognition sites on either strand (GGTCTC) to facilitate cloning. If BsaI sites are unavoidable, use an alternate type IIs restriction enzyme (BsmBI, SapI, BbsI) and modify the corresponding sites in the recipient plasmid (pUC19-BsaI), resistance marker donor plasmid (pGolden-Neo or pGolden-Hygro),....

Results

For the generation of ELAVL1 knockout lines, a robust antibody was available, so editing using single sgRNAs (Figure 1A, left) was performed, followed by dot immunoblot. Three sgRNAs were transfected independently to compare efficiencies and to rule out off target effects in the resulting clones. After collecting and blotting cell lysates from clonal populations onto two nitrocellulose membranes, the blots were probed for both ELAVL1 and PUM2 (as a normalizat.......

Discussion

The CRISPR/Cas9 system has allowed for efficient generation of stable genomic modifications, which provide a more consistent alternative to other transient manipulation methods. Here, we have presented two methods for rapid identification of CRISPR/Cas9 gene knockouts in mammalian cell lines. Both methods require little cellular material, so testing can be done in early stages of clonal culture, saving time and reagents. To increase the efficiency of both methods, we recommend testing multiple sgRNAs, as efficiencies var.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Competent E. coli cells
plasmid prep kit
pSpCas9(BB) plasmid	Addgene	42230	Ran et al. 2013, cloning sgRNAs
BbsI enzyme	ThermoFisher	FD1014	Ran et al. 2013, cloning sgRNAs
T7 DNA ligase	NEB	M0318L	Ran et al. 2013, cloning sgRNAs
Tango Buffer	ThermoFisher	BY5	Ran et al. 2013, cloning sgRNAs
PlasmidSafe exonuclease	Epicentre	E3105K	Ran et al. 2013, cloning sgRNAs
Q5 hot start high fidelity polymerase	NEB	M0494A	HA amplification
pUC19-BsaI			Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion
pGolden-Neo	Addgene	51422	Resistance cassette
pGolden-Hygro	Addgene	51423	Resistance cassette
BsaI enzyme	NEB	R3535S	Homology arm contruction
T7 DNA ligase	NEB	M0318L
PlasmidSafe exonuclease	Epicentre	E3105K
Toothpicks			bacterial PCR
Taq polymerase			bacterial colony PCR
HEK293 human cells
DMEM	Corning	10-013-CV
FBS	Corning	MT35010CV
Penicillin-Strep (opt.)	Gibco	15140-122
6 well plates	BioLite	12556004
TransIT-LTI Transfection Reagent	Mirus	MIR2300	for lipofection only
Opti-MEM	ThermoFisher	31985062	for lipofection only
G418 (Neomycin)	Sigma Aldrich	A1720-5G
Hygromycin	Sigma Aldrich	H3274-250MG
96 well plates	ThermoFisher	12556008
Passive Lysis Buffer, 5x	Promega
1x SDS loading buffer			recipe decribed in protocol
Nitrocellulose Membrane	Bio-Rad	162-0115
TBST			recipe decribed in protocol
Dehydrated milk
SuperSignal West Dura Extended Duration Substrate	ThermoFisher	34075	for HRP-conjugated secondary antibodies
Extracta DNA prep for PCR	Quantabio	95091-025
KOD polymerase	Novagen	71316