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%.
Gene Expression Omnibus
We thank all members of the Shendure and Trapnell labs for feedback on our manuscript and helpful discussions, particularly S. Srivatsan, G. Findlay, A. McKenna, R. Daza, B. Martin, M. Kircher, D. Cusanovich, X. Qiu, and V. Ramani. We thank J. Bloom and D. Fowler for discussions about lentivirus, and K. Han, J. Ousey, and M. Bassik for experimental advice and reagents for CRISPRi experiments. A.J.H. thanks Stella the cat for support. This work was supported by the following funding: NIH DP1HG007811 and UM1HG009408 (to J.S.), DP2HD088158 (to C.T.), and the W.M. Keck Foundation (to C.T. and J.S.). A.J.H. and M.J.G. are funded by the National Science Foundation Graduate Research Fellowship. J.L.M. is supported by the NIH Genome Training Grant (5T32HG000035) and the Cardiovascular Research Training Grant (4T32HL007828). C.T. is partly supported by an Alfred P. Sloan Foundation Research Fellowship. J.S. is an Investigator of the Howard Hughes Medical Institute.