CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells
Published: April 30, 2023
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…
Offenlegungen
The authors have nothing to disclose.
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 |
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CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells. J. Vis. Exp. (Pending Publication), e20984, doi: (2023).