dHyperCas12a enables multiplexed CRISPRi screens

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that the dCas12a system is highly effective for high-throughput screens. We use four different dCas12a repressors and a ~900-member library of crRNA arrays containing four crRNAs each to target the promoters of essential genes or cell surface negative controls. This fitness screen allows us to compare the activity of different dCas12a repressors across many genomic loci. Overall design: To perform a combinatorial screen at the PER1 locus, we created two crRNA array plasmid libraries, with each construct containing six crRNAs and a unique barcode. The PER1-targeting library contains ~9,000 crRNA arrays and the Non-targteing library contains ~10,000 crRNA arrays. Each plasmid library was sequenced twice. These libraries were then transduced into cells at low MOI and selected with puromycin for ten days. On day ten they were rreated with different concentrations of dexamethasone, and sorted on PER1 expression. Top and bottom 12% PER1-expressing cells were sorted and sequenced, as well as bulk unsorted cells (Input). To perform a fitness screen in A549 cells, we created two crRNA array plasmid libraries, containing the same spacers but either the As or Lb Cas12a direct repeat sequence, with each array construct containing four crRNAs. Each library contained ~900 crRNA arrays. These libraries were then transduced into A549 cells that stably express either dHyperLbCas12a-KRAB, dHyperLbCas12a-4xSID, dEnAsCas12a-KRAB, or dEnAsCas12a-4xSID repressors at low MOI (~0.2) and selected with puromycin. Three independent transductions were performed for each repressor cell line. Cells were passaged for three weeks post-transduction and cell pellets were collected at days 5, 7, 14, and 21 (post-transduction). crRNA array sequences were amplified from these cell pellets as well as the plasmid libraries and sequenced on the NextSeq2000.