Cargando…
A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18
CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10162949/ https://www.ncbi.nlm.nih.gov/pubmed/37159673 http://dx.doi.org/10.1016/j.crmeth.2023.100444 |
_version_ | 1785037791751569408 |
---|---|
author | Chen, Ye Meng, Lirong Lang, Bin Li, Lifang Liu, Jiashuo Wang, Yuyan Huang, Zheying Tian, Xun Tian, Rui Hu, Zheng |
author_facet | Chen, Ye Meng, Lirong Lang, Bin Li, Lifang Liu, Jiashuo Wang, Yuyan Huang, Zheying Tian, Xun Tian, Rui Hu, Zheng |
author_sort | Chen, Ye |
collection | PubMed |
description | CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis. |
format | Online Article Text |
id | pubmed-10162949 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-101629492023-05-07 A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 Chen, Ye Meng, Lirong Lang, Bin Li, Lifang Liu, Jiashuo Wang, Yuyan Huang, Zheying Tian, Xun Tian, Rui Hu, Zheng Cell Rep Methods Article CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis. Elsevier 2023-04-05 /pmc/articles/PMC10162949/ /pubmed/37159673 http://dx.doi.org/10.1016/j.crmeth.2023.100444 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Article Chen, Ye Meng, Lirong Lang, Bin Li, Lifang Liu, Jiashuo Wang, Yuyan Huang, Zheying Tian, Xun Tian, Rui Hu, Zheng A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title | A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title_full | A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title_fullStr | A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title_full_unstemmed | A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title_short | A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18 |
title_sort | cas12a ortholog with distinct ttna pam enables sensitive detection of hpv16/18 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10162949/ https://www.ncbi.nlm.nih.gov/pubmed/37159673 http://dx.doi.org/10.1016/j.crmeth.2023.100444 |
work_keys_str_mv | AT chenye acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT menglirong acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT langbin acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT lilifang acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT liujiashuo acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT wangyuyan acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT huangzheying acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT tianxun acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT tianrui acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT huzheng acas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT chenye cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT menglirong cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT langbin cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT lilifang cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT liujiashuo cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT wangyuyan cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT huangzheying cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT tianxun cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT tianrui cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 AT huzheng cas12aorthologwithdistinctttnapamenablessensitivedetectionofhpv1618 |