ABSTRACT The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user‐specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double‐strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several‐hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1–10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes. Biotechnol. Bioeng. 2015;112: 1060–1064. © 2014 Wiley Periodicals, Inc. The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. He and colleagues demonstrate that concurrent delivery of gRNAs designed to target two different sites in a human chromosome efficiently induce targeted deletion of the intervening genomic segment. Further, CRISPR/Cas9 mediated deletion of large genomic regions is more efficient than that induced by either ZFN or TALEN, offering an attractive strategy for investigating the functional consequences of large sequence deletions in cells and animals.