In vitro analysis of site specific nuclease selectivity by NGS

Vincent Brondani; Vincent Brondani

doi:10.3934/bioeng.2021020

AIMS Bioengineering

2021, Volume 8, Issue 4: 235-242. doi: 10.3934/bioeng.2021020

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In vitro analysis of site specific nuclease selectivity by NGS

Vincent Brondani ^,

GenAccurate, Colmar, France

Received: 30 July 2021 Accepted: 09 September 2021 Published: 16 September 2021

Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.
- Nucleases,
- in vitro cleavage,
- specificity,
- selectivity,
- NGS
Citation: Vincent Brondani. In vitro analysis of site specific nuclease selectivity by NGS[J]. AIMS Bioengineering, 2021, 8(4): 235-242. doi: 10.3934/bioeng.2021020

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Abstract

Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.

Abbreviations

BamHI

Bacillus amyloliquefaciens H I

Cas

CRISPR-associated protein

ClaI

Caryophanon latum I

Cpf1

CRISPR from Prevotella and Francisella 1

CRISPR

Clustered regularly interspaced short palindromic repeat

crRNA

CRISPR RNA

DSB

Double strand Break

DMEM

Dulbecco's modified Eagle Medium

DMSO

Dimethyl sulfoxide

EcoRI

E. coli RY13 I

FokI

Flavobacterium okeanokoites

MBP

Maltose binding protein

NGS

Next generation sequencing

PAM

Protospacer adjacent motif

SmaI

Serratia marcescens I

TALEN

transcription activator-like effector nucleases

ZFN

Zinc finger nuclease

Acknowledgments

The author is grateful to his previous colleagues from the Pasteur Institute to provide their help for the screening assay.

Conflict of interest

The author has declared no conflict of interest.

References

[1]	Kühnlein U, Linn S, Arber W (1969) Host specificity of DNA produced by Escherichia coli, XI. In vitro modification of phage fd replicative form. Proc Natl Acad Sci USA 63: 556-562. doi: 10.1073/pnas.63.2.556
[2]	Bibikova M, Carroll D, Segal DJ, et al. (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21: 289-297. doi: 10.1128/MCB.21.1.289-297.2001
[3]	Christian M, Cermak T, Doyle EL, et al. (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186: 757-761. doi: 10.1534/genetics.110.120717
[4]	Gasiunas G, Barrangou R, Horvath P, et al. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109: E2579-E2586. doi: 10.1073/pnas.1208507109
[5]	Jinek M, Chylinski K, Fonfara I, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821. doi: 10.1126/science.1225829
[6]	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163: 759-771. doi: 10.1016/j.cell.2015.09.038
[7]	Nishimasu H, Ran FA, Hsu PD, et al. (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156: 935-949. doi: 10.1016/j.cell.2014.02.001
[8]	Yamano T, Nishimasu H, Zetsche B, et al. (2016) Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165: 949-962. doi: 10.1016/j.cell.2016.04.003
[9]	Kim D, Kim J, Hur JK, et al. (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34: 863-868. doi: 10.1038/nbt.3609
[10]	Kamps-Hughes N, Quimby A, Zhu Z, et al. (2013) Massively parallel characterization of restriction endonucleases. Nucleic Acids Res 41: e119. doi: 10.1093/nar/gkt257
[11]	Murugan K, Seetharam AS, Severin AJ, et al. (2020) CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects: EDITORS'PICK: Cas12a nickase activities. J Biol Chem 295: 5538-5553. doi: 10.1074/jbc.RA120.012933

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