An alternative way for RNAi libraries to modify the mammalian genome on a large-scale may lie in the use of genome editing technologies such as the use of targeted endonucleases. Such a technique includes Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly-interspaced, short palindromic repeats/CRISPR-associated endonuclease cas9 (CRISPR/Cas9). CRISPR is a Cas9 endonuclease–based method for sequence-specific genome modification. It can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.
These technologies have proven highly useful for gene editing genomes of various species and the speed at which CRISPR techniques have been exploited has been incredibly rapid.
In January 2013 researchers at MIT, the Broad Institute, and Rockerfeller University first reported the new technique based on the CRISPR/Cas9 system, for precisely altering the genomes of living cells through gene addition or deletion. Researchers say the technology could offer an easy-to-use and more cost-effective way to engineer organisms that produce biofuels; to design animal models to study human disease; or to develop new therapies, among other potential applications.
Bioline reagents have been used to augment the workflow in these exciting gene editing technologies to elucidate CRISPR systems, including our SensiFAST™ range of Real-Time PCR Kits, as well as our performance PCR polymerases.
In the first examples of genes that mediate the inhibition of a CRISPR/Cas system, researchers from the University of Toronto identified five distinct ‘anti-CRISPR’ genes in the genomes of bacteriophages infecting Pseudomonas aeruginosa.
Bondy-Denomy, J et al., (2012). Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429-432.
Researchers from Minnesota University reported precise, high-frequency editing of 15 genes in pig, goat, and cattle genomes using CRISPR/Cas and TALENs technology. RFLP analysis of colonies were analysed by PCR using MyTaq Red Mix.
Wenfang, T.W. et al., (2013). Efficient nonmeiotic allele introgression in livestock using custom endonucleases. PNAS 110(41), 16526-16531.
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