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. Currently work is in progress to test combinatorial transcriptional repressor activities in our CRISPRi cell lines. Transcriptional activator domains have been cloned into lentiviral expression vectors carrying various CRISPR-Cas nucleases (Cas9 and Cas12a variants). HAP1 cells have been transduced with CRISPRa constructs (single and Cas9/Cas12a pairs) and nuclease expression has been assessed by western blotting. Editing efficiency of combined CRISPRa effectors have been tested by assessing expression of cell surface markers by flow cytometry. Current work is focused on modifying the CRISPRa effectors to enhance nuclease expression and hence genome editing efficiency.