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

Selkova, Polina LU ; Vasileva, Aleksandra ; Pobegalov, Georgii ; Musharova, Olga ; Arseniev, Anatolii ; Kazalov, Maksim ; Zyubko, Tatyana ; Shcheglova, Nataliia ; Artamonova, Tatyana and Khodorkovskii, Mikhail , et al. (2020) In RNA Biology 17(10). p.1472-1479
Abstract

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 5'-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... (More)

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 5'-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 22nd position of target strand, producing 3-5 nucleotide-long 5'-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 5'-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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publishing date
type
Contribution to journal
publication status
published
keywords
Base Sequence, Binding Sites, CRISPR-Associated Proteins/metabolism, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Computational Biology/methods, Gene Editing, Models, Molecular, Nucleic Acid Conformation, RNA Cleavage, RNA, Guide, CRISPR-Cas Systems/genetics, Recombinant Proteins
in
RNA Biology
volume
17
issue
10
pages
1472 - 1479
publisher
Taylor & Francis
external identifiers
  • pmid:32564655
  • scopus:85087342601
ISSN
1547-6286
DOI
10.1080/15476286.2020.1777378
language
English
LU publication?
no
id
0ed6c39d-c295-4c81-871d-458f6f2716f3
date added to LUP
2026-01-29 09:57:15
date last changed
2026-01-30 04:01:46
@article{0ed6c39d-c295-4c81-871d-458f6f2716f3,
  abstract     = {{<p>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 5'-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 22nd position of target strand, producing 3-5 nucleotide-long 5'-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 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.</p>}},
  author       = {{Selkova, Polina and Vasileva, Aleksandra and Pobegalov, Georgii and Musharova, Olga and Arseniev, Anatolii and Kazalov, Maksim and Zyubko, Tatyana and Shcheglova, Nataliia and Artamonova, Tatyana and Khodorkovskii, Mikhail and Severinov, Konstantin and Fedorova, Iana}},
  issn         = {{1547-6286}},
  keywords     = {{Base Sequence; Binding Sites; CRISPR-Associated Proteins/metabolism; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Computational Biology/methods; Gene Editing; Models, Molecular; Nucleic Acid Conformation; RNA Cleavage; RNA, Guide, CRISPR-Cas Systems/genetics; Recombinant Proteins}},
  language     = {{eng}},
  number       = {{10}},
  pages        = {{1472--1479}},
  publisher    = {{Taylor & Francis}},
  series       = {{RNA Biology}},
  title        = {{Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA}},
  url          = {{http://dx.doi.org/10.1080/15476286.2020.1777378}},
  doi          = {{10.1080/15476286.2020.1777378}},
  volume       = {{17}},
  year         = {{2020}},
}