Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection.
Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.
Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College.
Catherine J
CJ
Fricano-Kugler
Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College.
Michael R
MR
Williams
Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College.
Julia R
JR
Salinaro
Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College.
Meijie
M
Li
Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College; Bryan.W.Luikart@dartmouth.edu.
Bryan
B
Luikart
D000818:Animals; D001921:Brain / Q000502:physiology / Q000821:virology; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D059766:DNA End-Joining Repair; D055786:Gene Knockout Techniques / Q000379:methods*; D018014:Gene Transfer Techniques; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems; D013238:Stereotaxic Techniques
D000094704:RNA, Guide, CRISPR-Cas Systems