title: Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.
abstract: The clustered regularly interspaced short  [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.
authors.affiliation: From the Harvard Medical School.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Canver
authors.affiliation: From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
authors.affiliation: the Division of Hematology/Oncology, Boston Children's Hospital.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Dass
authors.affiliation: From the Harvard Medical School, the Division of Hematology, Brigham and Women's Hospital, and.
authors.forename: Yvette Y
authors.initials: YY
authors.lastname: Yien
authors.affiliation: From the Harvard Medical School, the Division of Hematology, Brigham and Women's Hospital, and.
authors.forename: Jacky
authors.initials: J
authors.lastname: Chung
authors.affiliation: the Division of Hematology, Brigham and Women's Hospital, and.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Masuda
authors.affiliation: From the Harvard Medical School, the Division of Hematology, Brigham and Women's Hospital, and.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maeda
authors.affiliation: From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute, the Division of Hematology, Brigham and Women's Hospital, and.
authors.forename: Barry H
authors.initials: BH
authors.lastname: Paw
authors.affiliation: From the Harvard Medical School, the Division of Hematology/Oncology, Boston Children's Hospital, the Department of Pediatric Oncology, Dana-Farber Cancer Institute, the Howard Hughes Medical Institute, Boston, Massachusetts 02115 Stuart_Orkin@dfci.harvard.edu.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000502:physiology*; D045744:Cell Line, Tumor; D017353:Gene Deletion; D023281:Genomics; D020071:Interspersed Repetitive Sequences; D051379:Mice; D008969:Molecular Sequence Data
keywords: CRISPR; Cas9; Gene Expression; Gene Knock-out; Gene Regulation; Genetics; Genome Engineering; Genomic Deletion; Genomics