Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus
Werner Neuhausser,
Hyung Oh,
Pierce Eggan,
Magdalena Angelova,
Rory Kirchner,
Kevin Eggan,
David Knipe
Affiliations
Werner Neuhausser
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USADepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, USA, Harvard Stem Cell Institute, Harvard University, Cambridge, USA
Hyung Oh
Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, USA
Pierce Eggan
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, USAHarvard Stem Cell Institute, Harvard University, Cambridge, USA
Magdalena Angelova
Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, USA
Rory Kirchner
Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, USA
Kevin Eggan
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, USAHarvard Stem Cell Institute, Harvard University, Cambridge, USA, Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, USA
David Knipe
Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, USA
The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.
