CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells

Abstract

Source: Merenda, A. et al., A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer. J. Vis. Exp. (2017).

This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.

Protocol

1. gRNA Design for the CRISPR-concatemer Vector NOTE: The aim of this section is to explain how to opt for the best targeting strategy and how to design gRNAs containing specific overhangs for the CRISPR-concatemer vector. Design gRNAs against the genes of interest using a CRISPR gRNA design tool of choice. See the Table of Materials for an example. NOTE: When targeting a pair of paralogous genes, although i…

Representative Results

Figure 1: Schematic Representation of the CRISPR-concatemer with 4 Cassettes. Scheme of the 4 gRNA-concatemer vectors with each 400 bp cassette containing a U6 promoter, two inverted repeated BbsI sites (also indicated as BB) and gRNA scaffold in this order. During the shuffling reaction, BbsI sites are replaced by gRNA fragments with matching overhangs and consequently lost. Bi…

Materials

Optimized CRISPR Design Tool	Feng Zhang group		CRISPR gRNA design tool; http://crispr.mit.edu/
Webcutter 2.0			restriction mapping tool; http://rna.lundberg.gu.se/cutter2/
T4 PNK (Polynucleotide Kinase)	New England Biolabs	M0201L
T4 DNA ligase buffer	New England Biolabs	M0202S
T7 DNA Ligase	New England Biolabs	M0318L
DTT (dithiothreitol)	Promega	P1171
ATP (adenosine triphosphate)	New England Biolabs	P0756S
FastDigest BbsI (BpiI)	Thermo Fisher	FD1014
Tango buffer (BSA-containing restriction enzyme buffer)	Thermo Fisher	BY5
BglII	New England Biolabs	R0144
EcoRI	New England Biolabs	R0101
Plasmid-safe exonuclease	Cambio	E3101K
Thermal cycler	Applied biosystems	4359659
10G competent E. coli bacteria	Cambridge Bioscience	60108-1
Advanced DMEM/F12(cell culture medium)	Invitrogen	12634-034
Glutamax (L-Glutamine)	100x Invitrogen	35050-068
HEPES 1 M (buffering agent)	Invitrogen	15630-056
Penicillin-streptomycin 100x	Invitrogen	15140-122
B27 supplement (Neuronal cell serum-free supplement) 50x	Invitrogen	17504-044
N2 supplement (Neuronal cell serum-free supplement) 100x	Invitrogen	17502-048
n-Acetylcysteine 500 mM	Sigma-Aldrich	A9165-5G
Mouse EGF 500 μg/mL	Invitrogen Biosource	PMG8043
Mouse Noggin 100 μg/mL	Peprotech	250-38
Nicotinamide 1 M	Sigma	N0636
R-Spondin conditioned medium	n.a.	n.a.	Produced in house from HEK293 cells, for details see Sato and Clevers 2013
Wnt conditioned medium	n.a.	n.a.	Produced in house from HEK293 cells, for details see Sato and Clevers 2014
Y-27632 10 μM	Sigma-Aldrich	Y0503-1MG
Standard BD Matrigel matrix	BD Biosciences	356231
48-well Plate	Greiner Bio One	677980
CHIR99021	Sigma-Aldrich	A3734-1MG
IWP-2	Cell Guidance Systems	SM39-10
TrypLE (recombinant protease)	Invitrogen	12605-010
Opti-MEM (reduced serum medium)	Life technologies	51985-034
Electroporation Cuvettes 2 mm gap	NepaGene	EC-002S
Low binding 15 mL tubes	Sigma-Aldrich	CLS430791
Bürker’s chamber	Sigma-Aldrich	BR719520-1EA
NEPA21 Super Electroporator	NepaGene	contact supplier
Protein LoBind tubes low binding	Thermo Fisher	10708704
BTXpress electroporation buffer	Harvard Apparatus	45-0805
DMSO (Dimethyl sulfoxide)	AppliChem	A3672