Author contributions K.C. and L.P. conceived the project, led the study, and wrote the software. K.C. analyzed experimental data. All authors contributed input on measurement and visualization of genome editing outcomes and provided input on the manuscript. Competing Interests At the time of manuscript preparation, J.M.G. was a consultant for Beam Therapeutics, and now is employed by Beam Therapeutics. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Endcadia, EpiLogic Therapeutics, Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.M.G. and J.K.J. are co-inventors on patents and patent applications that describe gene editing technologies. D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants and Beam Therapeutics, companies that use genome editing. Code availability.

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SHORT ABSTRACT CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. LONG ABSTRACT The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.