On the design of CRISPR-based single-cell molecular screens

CRISPR-based single-cell pooled screens that use linked barcodes suffer from lost sensitivity due to lentiviral template switching. The barcode-free CROP-seq design circumvents this problem. Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA–barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.