Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA

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Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA. / Paul, Bijoya; Chaubet, Loic; Verver, Dideke Emma; Montoya, Guillermo.

In: Nucleic Acids Symposium Series, Vol. 50, No. 9, 2022, p. 5208-5225.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Paul, B, Chaubet, L, Verver, DE & Montoya, G 2022, 'Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA', Nucleic Acids Symposium Series, vol. 50, no. 9, pp. 5208-5225. https://doi.org/10.1093/nar/gkab1272

APA

Paul, B., Chaubet, L., Verver, D. E., & Montoya, G. (2022). Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA. Nucleic Acids Symposium Series, 50(9), 5208-5225. https://doi.org/10.1093/nar/gkab1272

Vancouver

Paul B, Chaubet L, Verver DE, Montoya G. Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA. Nucleic Acids Symposium Series. 2022;50(9):5208-5225. https://doi.org/10.1093/nar/gkab1272

Author

Paul, Bijoya ; Chaubet, Loic ; Verver, Dideke Emma ; Montoya, Guillermo. / Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA. In: Nucleic Acids Symposium Series. 2022 ; Vol. 50, No. 9. pp. 5208-5225.

Bibtex

@article{4401574a539f405e852679d78212faf8,
title = "Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA",
abstract = "Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage lambda-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that lambda-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.",
keywords = "CPF1 ENDONUCLEASE, OPTICAL TWEEZERS, STRUCTURAL BASIS, STRANDED-DNA, GUIDE RNA, GENOME, COMPLEX, TARGET, SPECIFICITIES, CRISPR/CAS",
author = "Bijoya Paul and Loic Chaubet and Verver, {Dideke Emma} and Guillermo Montoya",
year = "2022",
doi = "10.1093/nar/gkab1272",
language = "English",
volume = "50",
pages = "5208--5225",
journal = "Nucleic acids symposium series",
issn = "0261-3166",
publisher = "Oxford University Press",
number = "9",

}

RIS

TY - JOUR

T1 - Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA

AU - Paul, Bijoya

AU - Chaubet, Loic

AU - Verver, Dideke Emma

AU - Montoya, Guillermo

PY - 2022

Y1 - 2022

N2 - Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage lambda-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that lambda-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.

AB - Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage lambda-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that lambda-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.

KW - CPF1 ENDONUCLEASE

KW - OPTICAL TWEEZERS

KW - STRUCTURAL BASIS

KW - STRANDED-DNA

KW - GUIDE RNA

KW - GENOME

KW - COMPLEX

KW - TARGET

KW - SPECIFICITIES

KW - CRISPR/CAS

U2 - 10.1093/nar/gkab1272

DO - 10.1093/nar/gkab1272

M3 - Journal article

C2 - 34951457

VL - 50

SP - 5208

EP - 5225

JO - Nucleic acids symposium series

JF - Nucleic acids symposium series

SN - 0261-3166

IS - 9

ER -

ID: 300908577