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Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA

Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a C...

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Autores principales: Selkova, Polina, Vasileva, Aleksandra, Pobegalov, Georgii, Musharova, Olga, Arseniev, Anatolii, Kazalov, Maksim, Zyubko, Tatyana, Shcheglova, Nataliia, Artamonova, Tatyana, Khodorkovskii, Mikhail, Severinov, Konstantin, Fedorova, Iana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549622/
https://www.ncbi.nlm.nih.gov/pubmed/32564655
http://dx.doi.org/10.1080/15476286.2020.1777378
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author Selkova, Polina
Vasileva, Aleksandra
Pobegalov, Georgii
Musharova, Olga
Arseniev, Anatolii
Kazalov, Maksim
Zyubko, Tatyana
Shcheglova, Nataliia
Artamonova, Tatyana
Khodorkovskii, Mikhail
Severinov, Konstantin
Fedorova, Iana
author_facet Selkova, Polina
Vasileva, Aleksandra
Pobegalov, Georgii
Musharova, Olga
Arseniev, Anatolii
Kazalov, Maksim
Zyubko, Tatyana
Shcheglova, Nataliia
Artamonova, Tatyana
Khodorkovskii, Mikhail
Severinov, Konstantin
Fedorova, Iana
author_sort Selkova, Polina
collection PubMed
description Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22(nd) position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.
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spelling pubmed-75496222020-10-27 Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA Selkova, Polina Vasileva, Aleksandra Pobegalov, Georgii Musharova, Olga Arseniev, Anatolii Kazalov, Maksim Zyubko, Tatyana Shcheglova, Nataliia Artamonova, Tatyana Khodorkovskii, Mikhail Severinov, Konstantin Fedorova, Iana RNA Biol Research Paper Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22(nd) position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments. Taylor & Francis 2020-06-21 /pmc/articles/PMC7549622/ /pubmed/32564655 http://dx.doi.org/10.1080/15476286.2020.1777378 Text en © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
spellingShingle Research Paper
Selkova, Polina
Vasileva, Aleksandra
Pobegalov, Georgii
Musharova, Olga
Arseniev, Anatolii
Kazalov, Maksim
Zyubko, Tatyana
Shcheglova, Nataliia
Artamonova, Tatyana
Khodorkovskii, Mikhail
Severinov, Konstantin
Fedorova, Iana
Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title_full Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title_fullStr Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title_full_unstemmed Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title_short Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA
title_sort position of deltaproteobacteria cas12e nuclease cleavage sites depends on spacer length of guide rna
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549622/
https://www.ncbi.nlm.nih.gov/pubmed/32564655
http://dx.doi.org/10.1080/15476286.2020.1777378
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