Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Richardson, C.D. et al. Nature Biotech (2016) http://www.ncbi.nlm.nih.gov/pubmed/26789497

After DNA cleavage with CRISPR/Cas9, cleaved DNA is repaired by one of two mechanisms.  Non-homologous end joining (NHEJ) is an error-prone repair mechanism that usually results in a non-functional gene product and is commonly used by researchers to knockout targeted genes.  Homology-directed repair (HDR) is the insertion of DNA sequences that have ends homologous to the cleavage sight and can be used for precise sequence replacement/addition to the genome, however the efficiency of HDR is extremely low.  Through the use of in vitro kinetic studies, Richardson et al. determined that Cas9 has a very slow rate of dissociation (~6 hours) with targeted DNA, however the non-target strand is released much faster with Cas9 remaining bound to only the targeted strand.  By rationally designing single-stranded DNA donor templates complementary to the non-target strand Richardson et al. increased the efficiency of HDR in human cell lines up to 60% when using eight wild-type or nickase variants of Cas9, and up to 0.7% when using a catalytically dead Cas9 that binds, but cannot cleave DNA.

Author: Advanced Analytical

Advanced Analytical Technologies, Inc. (AATI) simplifies complex genomics workflows to accelerate research and discovery in pharmaceuticals, life science, biofuels, biotechnology and healthcare.

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