Fig. 2.

A guide RNA–target pair strategy for high-throughput screen of gRNA activity in human cells. a Schematic diagram of the guide RNA–target pair strategy for gRNA activity test. A lentiviral vector contains a mU6 promoter, and a guide RNA–target pair. The vector was used to transduce cells expressing Cas9 nucleases. Indels would be induced at integrated targets by the corresponding gRNAs. b Schematic diagram of design and high-throughput screen of a gRNA library. A library of 80,263 guide RNA–target pairs was designed and synthesized by microarray. Oligonucleotides were PCR-amplified and cloned into lentiviral vectors by Gibson assembly. The library was packed into viruses and transduced into cells expressing Cas9 nucleases for genome editing. The integrated target sites were PCR-amplified for deep-sequencing analysis. c The Pearson correlation of indel frequency among different experiment repeats. d The distribution of gRNA activity for the three Cas9 nucleases