Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

ABSTRACT: Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including huma

SUBMITTER: Kelkar A 

PROVIDER: S-EPMC6952177 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature