Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies novel HIV-1 restriction factors in Jurkat 2D10 cells.

A. A diagram showing the procedure of REACT that involves repeated cycles of CRISPRi, FACS-selection of GFP+ cells, and subcloning of the sgRNA library to enrich the sgRNA sequences that target host HIV-1 restriction factors. The 2D10-based TetOn mCherry-dCas9-KRAB cell line (called 2D10-CRISPRi) was used. B. Representative FACS plots of 2D10-CRISPRi cells non-transduced or transduced by original or enriched sgRNA libraries. The cells were first treated with either DMSO (Dox-) or doxycycline (Dox+) to induce the expression of the KRAB-dCas9-HA-P2A-mCherry fusion protein and then selected by FACS for the GFP+ cells harboring activated HIV-1. C. The round 4-enriched and original sgRNA libraries were subjected to high throughput sequencing and the fold of enrichment for each sgRNA was calculated based on its reads per million in the round 4-enriched library divided by those in the original library and presented on a scatter plot, with the 7 most significantly enriched sequences highlighted in green. D. Shown are the sequences of the 7 sgRNA highlighted in C and their target genes.