Genome-wide CRISPR-Cas screen identifies CRNKL-1 as a nuclear retention factor of HIV-1 unspliced RNA

ABSTRACT: The HIV-1 regulatory Rev protein binds to unspliced or incompletely-spliced HIV-1 RNAs and thereby targets these RNAs for nuclear export and translation. Without Rev, these intron-retaining RNAs are trapped in the nucleus. To identify cellular factors involved in nuclear trapping of intron-retaining HIV-1 RNAs, a genome-wide lentiviral CRISPR/Cas knockout vector library was screened for enhanced reporter gene expression from the unspliced HIV-1 RNA. Reporter cell line establishment A dual-color proviral reporter plasmid, HIV-dual-GT, was first constructed. gag is ligated by a BFP; nef is replaced by a DsRed; rev, pol and other accerssory genes are mutatd by point mutations. tat remains functional for active transcription. Jurkat CD4 T cells were transduced by this proviral vector. Two cell clones, designated J-dual#3 and 6, which contain the viral reporter genome and thefere express DsRed were isolated and characterized. Genome-wide CRISPR/Cas screen A pooled human genome-wide knockout library, GeCKO v2, was purchased from Addgene and used to transduce the J-dual#3, J-dual#6 and parental Jurkat at an MOI of 2. 4 days later, BFP-postive cells from J-dual#3 and J-dual#6 populations were sorted. Selected cells from the two reporter clones and unselcted Jurkat cells (CTRL) were lysed. sgRNA sequences were amplified, indexed and subjected to Illumina sequencing. Enriched sampled using MiSeq, unselected CTRL using HiSeq 2500. NGS raw counts were anayzed by the MAGeCK tool.

SUBMITTER: Han Xiao 

PROVIDER: S-BSST243 | biostudies-other |

REPOSITORIES: biostudies-other