(627f) Doxycycline Dependent Self-Inactivation of CRISPR-SpCas9 to Temporally Regulate on- and Off-Target Editing | AIChE

(627f) Doxycycline Dependent Self-Inactivation of CRISPR-SpCas9 to Temporally Regulate on- and Off-Target Editing


Neelamegham, S. - Presenter, University at Buffalo, State University of New York
Kelkar, A., State University of New York-Buffalo
Zhu, Y., State University of New York-Buffalo
Groth, T., State University of New York-Buffalo
Stolfa, G., State University of New York-Buffalo
Stablewski, A., Roswell Park Cancer Institute
Nemeth, M., Roswell Park Cancer Institute
Singhi, N., Roswell Park Cancer Institute
The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system is widely used for genome editing due to its simplicity, versatility and efficiency. Among the endonucleases used in such applications, the Streptococcus pyogenes Cas9 (‘Cas9’) is common as it efficiently induces site-directed double stranded breaks to enable either site-specific indel (insertion-deletion) formation during non-homologous end joining (NHEJ) or nucleotide insertion during homology-dependent repair (HDR). While efficient, a number of studies suggest that CRISPR/Cas9 applications can have unintended off-target effects (OTEs). Such OTE is particularly unacceptable in the context of human clinical applications. Since Cas9 tolerates a few base mismatches, these can also accumulate in forward genetic screen applications where sgRNA and Cas9 are stably integrated into the chromosome. In our studies, also, we observed using exome sequencing of cells treated with a panel of lentiviral guide RNA 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 developed 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 human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion. 2. Genome-wide lentiviral sgRNA library applications 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 mediated editing 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 studies that aim to reduce off-target genome editing.