A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6⍺

Activating transcription factor 6 alpha (ATF6⍺) is one of the three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its significant involvement in long-term ER stress adaption, regulation of ATF6⍺ signalling is still poorly understood, possibly because its activation involves Golgi and nucleus trafficking. Here, we have generated a dual CHO-K1 ATF6⍺/IRE1⍺ reporter cell line to perform an unbiased genome-wide CRISPR/Cas9 mutagenesis screen, in the presence and absence of ER stress, to systematically profile genetic factors that specifically contribute to ATF6⍺ signalling. Anticipated and new candidate genes that regulate ATF6⍺ activation were discovered. Among these, calreticulin (CRT), a key ER luminal chaperone, emerged as a selective repressor molecule of ATF6⍺ signalling. Cells lacking CRT constitutively activated a BiP::sfGFP ATF6⍺-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6⍺. Purified CRT interacts with the luminal domain of ATF6⍺ in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6⍺ in repressing IRE1⍺ activity basally and overexpression of CRT reversed phenotype. Our data indicate that CRT, in addition to its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to maintain selective regulation of the UPR. Examination of genomic DNA, pooled from sorted cells in different bins (based on ATF6::sfGFP and XBP1s::mCherry reporter signals) at different stages of the phenotypic enrichment process and from unsorted control cells, was subjected to high-throughput sequencing and MAGeCK bioinformatics analysis. In total 16 samples were examinated.