Berk, C. | Search bioRxiv

bioRxiv2017

Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

The CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.

molecular biology↗

bioRxiv2017

Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases

In many prokaryotes, type III CRISPR-Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

biochemistry↗