Multi-target CRISPR for high-throughput studies of double-strand break repair and genetic barcoding at endogenous sites

CRISPR has revolutionized biology by enabling highly versatile and precise genome editing. In the canonical application of CRISPR, a guide RNA (gRNA) is rationally designed to direct the nuclease to an individual genomic locus. However, for many other applications of CRISPR, it is desirable to target a large number of genomic locations at once. Here, we demonstrate efficient multiplexing of CRISPR using multi-target gRNAs (mgRNAs). By targeting mutations of the Alu repetitive element dispersed throughout the genome, mgRNAs introduce a user-defined number of on-target double-strand breaks (DSBs).