CRISPR-Cas9 cleavage efficiency correlates strongly with targetsgRNA folding stability: From physical mechanism to off-target assessment

Scientific Reports

Volumn 7, Issue 1, 2017, Pages

  Xu, Xiaojun ^a   Duan, Dongsheng ^b,c,d   Chen, Shi Jie ^a  

^a University of Missouri   (United States)

^b School of Medicine   (United States)

^c UNIVERSITY OF MISSOURI   (United States)

^d UNIVERSITY OF MISSOURI   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT; CORRELATION COEFFICIENT; DNA RNA HYBRIDIZATION; GENOME; MECHANICS; CHEMISTRY; CONFORMATION; CRISPR CAS SYSTEM; GENE EDITING; GENETICS; METABOLISM; MOLECULAR MODEL; MUTATION; STATISTICAL MODEL;

DNA; ENDONUCLEASE; GUIDE RNA;

CRISPR-CAS SYSTEMS; DNA; ENDONUCLEASES; GENE EDITING; MODELS, MOLECULAR; MODELS, STATISTICAL; MUTATION; NUCLEIC ACID CONFORMATION; RNA, GUIDE;

EID: 85020293176     PISSN: None     EISSN: 20452322     Source Type: Journal    
DOI: 10.1038/s41598-017-00180-1     Document Type: Article

References (36)

1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121), 819-823 (2013).
2. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339(6121), 823-826 (2013).
3. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6), 1262-1278 (2014).
4. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213), 1258096 (2014).
5. Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 16(5), 299-311 (2015).
6. Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet 17(5), 300-312 (2016).
7. Xiong, X., Chen, M., Lim, W. A., Zhao, D. & Qi, L. S. CRISPR/Cas9 for Human Genome Engineering and Disease Research. Annu Rev Genomics Hum Genet 17, 131-154 (2016).
8. Barrangou, R. & Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 34(9), 933-941 (2016).
9. Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17(1), 148 (2016).
10. Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351(6271), 403-407 (2016).
11. Wu, X. et al. Genome-wide bound of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol. 32(7), 670-676 (2014).
12. Szczelkun, M. D. et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc Natl Acad Sci USA 111(27), 9798-9803 (2014).
13. Josephs, E. A. et al. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target bound and cleavage. Nucleic Acids Res 43(18), 8924-8941 (2015).
14. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351(6268), 84-88 (2016).
15. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156(5), 935-949 (2014).
16. Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513(7519), 569-573 (2014).
17. Singh, R., Kuscu, C., Quinlan, A., Qi, Y. & Adli, M. Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucl Acids Res 43(18), e118 (2015).
18. Stemmer, M., Thumberger, T., Del Sol Keyer, M., Wittbrodt, J. & Mateo, J. L. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10(4), e0124633 (2015).
19. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 31(9), 827-832 (2013).
20. Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 32(12), 1262-1267 (2014).
21. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 34(2), 184-191 (2016).
22. Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42(11), 7473-7485 (2014).
23. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-1183 (2013).
24. Liu, Y. et al. Targeting cellular mRNAs translation by CRISPR-Cas9. Sci Rep. 6, 29652 (2016).
25. SantaLucia, J. Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA. 95(4), 1460-1465 (1998).
26. Zhang, W. & Chen, S.-J. RNA hairpin-folding kinetics. Proc Natl Acad Sci USA 99(4), 1931-1936 (2002).
27. Xu, X. & Chen, S.-J. Kinetic mechanism of conformational switch between bistable RNA hairpins. J Am Chem Soc. 134(30), 12499-12507 (2012).
28. Sugimoto, N. et al. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34(35), 11211-11216 (1995).
29. Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 32(7), 677-683 (2014).
30. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 33(2), 187-197 (2015).
31. Rastrigin, L. A. The convergence of the random search method in the extremal control of a many-parameter system. Autom Remote Control 24, 1337-1342 (1963).
32. Solis, F. J. & Wets, R. J. B. Minimization by random search techniques. Math Oper Res. 6(1), 19-30 (1981).
33. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 32(3), 279-284 (2014).
34. Zhang, J. P. et al. Different effects of sgRNA length on CRISPR-mediated gene knockout efficiency. Sci Rep. 6, 28566 (2016).
35. Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345(6201), 1184-1188 (2014).
36. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523(7561), 481-485 (2015).