Development of a CRISPR‐Cas9 Based Luciferase Turn‐On System as Nonhomologous End Joining Pathway Reporter

Abstract

There is a need of a non‐homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR‐Cas9 based luciferase turn‐on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading‐frame shift and a pre‐stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line. Then, a CRISPR‐Cas9 plasmid expressing a guide RNA targeting luciferase coding region was introduced into the reporter cell line to generate NHEJ‐associated indel to restore the reading frame and subsequently turn on the bioluminescent signal. We observed over three‐thousand fold increase in signal after CRISPR‐Cas9 vector transfection. Different known chemical inhibitors of the NHEJ pathway, such as NU7441, KU0060648, and KU55933, could significantly inhibit the bioluminescent signal generated by CRISPR‐Cas9 targeting. In addition, we validated our system by high throughput sequencing.

Mechanism-guided engineering of a minimal biological particle for genome editing

“…[13][14][15] We produced EDVs packaging Cas9 RNPs that target a prematurely truncated luciferase reporter gene (C205ATC). 16 HIV-1 lentiviral vectors packaging a transgene encoding Cas9 enzymes and the same guide RNA were used as a positive control. The particles were incubated with HEK-293T cells expressing the truncated luciferase reporter in either the presence or absence of the inhibitors.…”