Combinatorial CRISPR transcriptional perturbation screening platform

Division Of Basic Sciences - Nci

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Abstract

Transcriptional repressor domains have been cloned into lentiviral expression vectors carrying various CRISPR-Cas nucleases (Cas9 and Cas12a variants). HAP1 and RPE1 cells have been transduced with CRISPRi constructs (single and Cas9/Cas12a pairs) and nuclease expression has been verified by western blotting. Editing efficiency of individual CRISPRi effectors have been verified by assessing expression of cell surface markers by flow cytometry. Transcriptional activator domains are also beeing cloned into lentiviral expression vectors carrying various CRISPR-Cas nucleases (Cas9 and Cas12a variants) and will be tested in HAP1 cells by flow cytometry and qRT-PCR. After observing strong cell line editing and nuclease expression variation we are currently focusing on modifying the CRISPRa effectors to enhance nuclease expression and hence genome editing efficiency.