PRJNA557186
Whole Exome Sequencing of Doxycycline-Inducible Cas9-harboring HEK293
Exome 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 activitywould be beneficial. To address this need, this manuscript describes a 'self-inactivating CRISPR (SiC)'system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease ina doxycycline dependent manner. This enables defined, temporal control of Cas9 activity in any cell-typeand 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 geneticknockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification ofglycogenes regulating leukocyte-endothelial cell adhesion. 2. Genome-wide lentiviral sgRNA libraryapplications where Cas9 activity is ablated using SiC after allowing pre-determined editing times. Thus,stable knockout cell pools are created for functional screens. 3. Temporal control of Cas9 mediatedediting of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall,SiC enables temporal control of gene editing and may be applied in diverse application including studiesthat aim to reduce off-target genome editing.