CRISPR Knockout

CRISPR-mediated gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

The KO technique is essentially the opposite of a gene knockin. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively. One needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a method for genome editing that contains a guide RNA complexed with a Cas9 protein [1]. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing, as opposed to the time consuming assembly of constructs required by zinc-fingers or TALENs [2]. The coupled Cas9 will cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.

A conditional knockout allows gene deletion in a tissue in a time specific manner. This is required in place of a gene knockout if the null mutation would lead to embryonic death [3]. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knockout. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.

CRISPR Knockout Contains The Following Sections

CRISPR Knockout Related References

1. Gaj, Thomas et al. "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering". Trends in Biotechnology. 31 (7): 397–405.
2. Ni et al. (2014-09-04). "Efficient Gene Knockout in Goats Using CRISPR/Cas9 System". PLOS ONE. 9 (9): e106718.
3. Le, Yunzheng; Sauer, Brian (2001-03-01). "Conditional gene knockout using cre recombinase". Molecular Biotechnology. 17 (3): 269–275.