title: Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.
abstract: The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.
authors.affiliation: RIKEN Brain Science Institute, Wako, Saitama, Japan.
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Liu
authors.affiliation: RIKEN Brain Science Institute, Wako, Saitama, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Homma
authors.affiliation: RIKEN Brain Science Institute, Wako, Saitama, Japan.|Harvard College, Cambridge, Massachusetts 02138, United States.
authors.forename: Jamasb
authors.initials: J
authors.lastname: Sayadi
authors.affiliation: Department of Computer Science, University of British Columbia, Vancouver, Canada.
authors.forename: Shu
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo, Tokyo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohashi
authors.affiliation: RIKEN Brain Science Institute, Wako, Saitama, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takumi
mesh_terms: D001482:Base Composition; D064113:CRISPR-Cas Systems; D000072669:Gene Editing; D018390:Gene Targeting; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
references.citation: Nat Rev Genet. 2015 May;16(5):299-311
references.pmid: 25854182
references.citation: Genome Biol. 2012;13(8):418
references.pmid: 22889292
references.citation: Nat Biotechnol. 2014 Jul;32(7):670-6
references.pmid: 24752079
references.citation: Mol Autism. 2013 Oct 03;4(1):36
references.pmid: 24090431
references.citation: Science. 2014 Nov 28;346(6213):1258096
references.pmid: 25430774
references.citation: Algorithms Mol Biol. 2011 Nov 24;6:26
references.pmid: 22115189
references.citation: Cell Rep. 2014 Nov 6;9(3):1151-62
references.pmid: 25437567
references.citation: Nature. 2012 Sep 6;489(7414):75-82
references.pmid: 22955617
references.citation: PLoS One. 2014;9(5):e98186
references.pmid: 24873830
references.citation: Mol Cell. 2015 May 21;58(4):568-74
references.pmid: 26000842
references.citation: Genome Res. 2015 Aug;25(8):1147-57
references.pmid: 26063738
references.citation: Cell. 2014 Feb 27;156(5):935-49
references.pmid: 24529477
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references.pmid: 22949671
references.citation: Nat Methods. 2015 Sep;12(9):823-6
references.pmid: 26167643
references.citation: Mol Cell. 2010 May 28;38(4):576-89
references.pmid: 20513432
references.citation: Science. 2014 Jan 3;343(6166):80-4
references.pmid: 24336569
references.citation: Cell Stem Cell. 2014 Nov 6;15(5):643-52
references.pmid: 25517468
references.citation: Nature. 2014 Dec 11;516(7530):263-6
references.pmid: 25274302
references.citation: Nucleic Acids Res. 2014 Jun;42(10):6091-105
references.pmid: 24728998
references.citation: Nature. 2014 Sep 25;513(7519):569-73
references.pmid: 25079318
references.citation: Cell. 2014 Jun 5;157(6):1262-78
references.pmid: 24906146
references.citation: Nat Biotechnol. 2013 Sep;31(9):827-32
references.pmid: 23873081
references.citation: Nat Methods. 2015 Oct;12(10):982-8
references.pmid: 26322839
references.citation: Nucleic Acids Res. 2014 Oct 29;42(19):e147
references.pmid: 25122746
references.citation: Nat Rev Microbiol. 2011 Jun;9(6):467-77
references.pmid: 21552286
references.citation: Science. 2013 Feb 15;339(6121):819-23
references.pmid: 23287718
references.citation: Nature. 2015 Jan 29;517(7536):583-8
references.pmid: 25494202
references.citation: Nat Biotechnol. 2015 May;33(5):510-7
references.pmid: 25849900
references.citation: Cell. 2013 Jul 18;154(2):442-51
references.pmid: 23849981
references.citation: RNA. 2010 Jun;16(6):1096-107
references.pmid: 20418358
references.citation: Nat Biotechnol. 2013 Sep;31(9):839-43
references.pmid: 23934178
references.citation: Nucleic Acids Res. 2015 Oct 15;43(18):e118
references.pmid: 26032770
references.citation: Nat Biotechnol. 2014 Dec;32(12):1262-7
references.pmid: 25184501
references.citation: Science. 2014 Mar 14;343(6176):1247997
references.pmid: 24505130
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249
references.citation: Nat Methods. 2014 Jan;11(1):28
references.pmid: 24524135