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Development of the Cas12a-based microdeletion and microinsertion detection system
- Authors: Chirinskaite A.V.1, Zelinsky A.A.1, Sopova J.V.1,2, Leonova E.I.1
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Affiliations:
- Saint Petersburg State University
- Vavilov Institute of General Genetics
- Issue: Vol 21 (2023): Спецвыпуск
- Pages: 20-21
- Section: Genetically modified organism. The Нistory, Achivements, Social and Environmental Riscs
- URL: https://journals.eco-vector.com/ecolgenet/article/view/568454
- DOI: https://doi.org/10.17816/ecogen568454
- ID: 568454
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Abstract
CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease from Lachnospiraceae bacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNA in vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.
In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice.
This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)
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CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease from Lachnospiraceae bacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNA in vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.
In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice.
This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)
About the authors
Angelina V. Chirinskaite
Saint Petersburg State University
Email: ChirinskaiteA@yandex.ru
ORCID iD: 0000-0002-7466-0680
junior researcher, center for transgenesis and genome editing
Russian Federation, Saint PetersburgAndrew A. Zelinsky
Saint Petersburg State University
Email: andrew_zelinsky@mail.ru
ORCID iD: 0000-0003-2068-3024
m. sci. (biology), researcher, laboratory of amyloid biology
Russian Federation, Saint PetersburgJulia V. Sopova
Saint Petersburg State University; Vavilov Institute of General Genetics
Email: sopova@hotmail.com
ORCID iD: 0000-0002-7825-273X
cand. sci. (biol.), leading researcher, center for transgenesis and genome editing
Russian Federation, Saint PetersburgElena I. Leonova
Saint Petersburg State University
Author for correspondence.
Email: 1102.elena@gmail.com
ORCID iD: 0000-0002-0236-3302
phd, head, center for transgenesis and genome editing
Russian Federation, Saint PetersburgReferences
- Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360(6387):439–444. doi: 10.1126/science.aaq0179
- Swarts DC, Jinek M. Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing. Wiley Interdiscip Rev RNA. 2018;9(5):e1481. doi: 10.1002/wrna.1481.
- Misiurina MA, Chirinskaite AV, Fotina AS, et al. New PAM Improves the single-base specificity of crRNA-guided LbCas12a nuclease. Life. 2022;12(11):1927. doi: 10.3390/life12111927
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