CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes Without RNA/DNA Complementarity

Overview

Journal ACS Omega

Publisher American Chemical Society

Specialty Chemistry

Date 2019 Oct 29

PMID 31656887

Citations 8

Authors

Wei Jiang

Jaideep Singh

Aleique Allen

Yue Li

Venkatesan Kathiresan

Omair Qureshi

Narin Tangprasertchai

Xiaojun Zhang

Hari Priya Parameshwaran

Rakhi Rajan

Peter Z Qin

Affiliations

Soon will be listed here.

Abstract

Cas12a (also known as "Cpf1") is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.

Citing Articles

PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.

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References

Kim D, Kim J, Hur J, Been K, Yoon S, Kim J . Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016; 34(8):863-8. DOI: 10.1038/nbt.3609. View

Pattanayak V, Lin S, Guilinger J, Ma E, Doudna J, Liu D . High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31(9):839-43. PMC: 3782611. DOI: 10.1038/nbt.2673. View

OGeen H, Henry I, Bhakta M, Meckler J, Segal D . A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2015; 43(6):3389-404. PMC: 4381059. DOI: 10.1093/nar/gkv137. View

Li S, Cheng Q, Liu J, Nie X, Zhao G, Wang J . CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 2018; 28(4):491-493. PMC: 5939048. DOI: 10.1038/s41422-018-0022-x. View

Swarts D, van der Oost J, Jinek M . Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Mol Cell. 2017; 66(2):221-233.e4. PMC: 6879319. DOI: 10.1016/j.molcel.2017.03.016. View

Mekler V, Minakhin L, Severinov K . Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation. Proc Natl Acad Sci U S A. 2017; 114(21):5443-5448. PMC: 5448204. DOI: 10.1073/pnas.1619926114. View

Kleinstiver B, Pattanayak V, Prew M, Tsai S, Nguyen N, Zheng Z . High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016; 529(7587):490-5. PMC: 4851738. DOI: 10.1038/nature16526. View

Kleinstiver B, Sousa A, Walton R, Tak Y, Hsu J, Clement K . Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol. 2019; 37(3):276-282. PMC: 6401248. DOI: 10.1038/s41587-018-0011-0. View

Chen J, Ma E, Harrington L, Da Costa M, Tian X, Palefsky J . CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018; 360(6387):436-439. PMC: 6628903. DOI: 10.1126/science.aar6245. View

10.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View

11.

Stella S, Alcon P, Montoya G . Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. 2017; 546(7659):559-563. DOI: 10.1038/nature22398. View

12.

Sternberg S, LaFrance B, Kaplan M, Doudna J . Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015; 527(7576):110-3. PMC: 4859810. DOI: 10.1038/nature15544. View

13.

Abudayyeh O, Gootenberg J, Konermann S, Joung J, Slaymaker I, Cox D . C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353(6299):aaf5573. PMC: 5127784. DOI: 10.1126/science.aaf5573. View

14.

Dong D, Ren K, Qiu X, Zheng J, Guo M, Guan X . The crystal structure of Cpf1 in complex with CRISPR RNA. Nature. 2016; 532(7600):522-6. DOI: 10.1038/nature17944. View

15.

Tang X, Lowder L, Zhang T, Malzahn A, Zheng X, Voytas D . A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants. 2017; 3:17018. DOI: 10.1038/nplants.2017.18. View

16.

Kleinstiver B, Tsai S, Prew M, Nguyen N, Welch M, Lopez J . Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol. 2016; 34(8):869-74. PMC: 4980201. DOI: 10.1038/nbt.3620. View

17.

Chen B, Gilbert L, Cimini B, Schnitzbauer J, Zhang W, Li G . Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 2013; 155(7):1479-91. PMC: 3918502. DOI: 10.1016/j.cell.2013.12.001. View

18.

Strohkendl I, Saifuddin F, Rybarski J, Finkelstein I, Russell R . Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol Cell. 2018; 71(5):816-824.e3. PMC: 6679935. DOI: 10.1016/j.molcel.2018.06.043. View

19.

Koonin E, Makarova K, Zhang F . Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017; 37:67-78. PMC: 5776717. DOI: 10.1016/j.mib.2017.05.008. View

20.

Knott G, Doudna J . CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361(6405):866-869. PMC: 6455913. DOI: 10.1126/science.aat5011. View