Abstract
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.
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