High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

Proceedings of the National Academy of Sciences of the United States of America

Volumn 114, Issue 21, 2017, Pages 5461-5466

  Boyle, Evan A ^a   Andreasson, Johan O L ^a   Chircus, Lauren M ^a   Sternberg, Samuel H ^b,e   Wu, Michelle J ^c   Guegler, Chantal K ^b,f   Doudna, Jennifer A ^b,d   Greenleaf, William J ^a  

^a STANFORD UNIVERSITY   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

^d LAWRENCE BERKELEY NATIONAL LABORATORY   (United States)

^e Caribou Biosciences Inc   (United States)

^f MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

CRISPR; DNA; Kinetics; Molecular biophysics; Sequencing

Indexed keywords

GUIDE RNA; NUCLEASE DEAD CAS9; NUCLEOTIDE; PEPTIDES AND PROTEINS; UNCLASSIFIED DRUG; BACTERIAL PROTEIN; CAS9 ENDONUCLEASE STREPTOCOCCUS PYOGENES; DNA; ENDONUCLEASE;

ARTICLE; BINDING AFFINITY; BINDING ASSAY; BINDING KINETICS; BIOCHEMICAL ANALYSIS; CELL SURFACE; CRISPR CAS SYSTEM; DISSOCIATION; DNA DENATURATION; DNA SEQUENCE; DNA TEMPLATE; ENZYME KINETICS; FLUORESCENCE; GEL MOBILITY SHIFT ASSAY; HIGH THROUGHPUT SEQUENCING; HYBRIDIZATION; PRIORITY JOURNAL; PROTEIN DNA BINDING; PROTEIN DNA INTERACTION; THERMODYNAMICS; HIGH THROUGHPUT SCREENING; METABOLISM;

BACTERIAL PROTEINS; DNA; ENDONUCLEASES; HIGH-THROUGHPUT SCREENING ASSAYS; RNA, GUIDE;

EID: 85019580103     PISSN: 00278424     EISSN: 10916490     Source Type: Journal    
DOI: 10.1073/pnas.1700557114     Document Type: Article

References (29)

1. Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569-573.
2. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733-740.
3. Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227-264.
4. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62-67.
5. Szczelkun MD, et al. (2014) Direct observation of R-loop formation by single RNAguided Cas9 and Cascade effector complexes. Proc Natl Acad Sci USA 111:9798-9803.
6. Duan J, et al. (2014) Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell Res 24:1009-1012.
7. Fu BXH, Hansen LL, Artiles KL, Nonet ML, Fire AZ (2014) Landscape of target:guide homology effects on Cas9-mediated cleavage. Nucleic Acids Res 42:13778-13787.
8. Pattanayak V, et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839-843.
9. Wu X, et al. (2014) Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32:670-676.
10. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M (2014) Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32:677-683.
11. Hsu PD, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827-832.
12. Sternberg SH, LaFrance B, Kaplan M, Doudna JA (2015) Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527:110-113.
13. Jiang F, et al. (2016) Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351:867-871.
14. O'Geen H, Henry IM, Bhakta MS, Meckler JF, Segal DJ (2015) A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res 43:3389-3404.
15. Xu H, et al. (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25:1147-1157.
16. Nutiu R, et al. (2011) Direct measurement of DNA affinity landscapes on a highthroughput sequencing instrument. Nat Biotechnol 29:659-664.
17. Buenrostro JD, et al. (2014) Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat Biotechnol 32:562-568.
18. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233-239.
19. Zhang Y, et al. (2014) Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep 4:5405.
20. Jinek M, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816-821.
21. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279-284.
22. Josephs EA, et al. (2016) Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res 44: 2474.
23. Singh D, Sternberg SH, Fei J, Doudna JA, Ha T (2016) Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat Commun 7:12778.
24. Farasat I, Salis HM (2016) A biophysical model of CRISPR/Cas9 activity for rational design of genome editing and gene regulation. PLOS Comput Biol 12:e1004724.
25. Fu Y, et al. (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822-826.
26. Kleinstiver BP, et al. (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490-495.
27. Oakes BL, et al. (2016) Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nat Biotechnol 34:646-651.
28. Slaymaker IM, et al. (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84-88.
29. Nihongaki Y, Kawano F, Nakajima T, Sato M (2015) Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol 33:755-760.