Tags

Type your tag names separated by a space and hit enter

Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA.
RNA Biol. 2020 Jun 21 [Online ahead of print]RB

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 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.

Authors+Show Affiliations

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences , Moscow, Russia.Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences , Moscow, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Skolkovo Institute of Science and Technology, Center of Life Sciences , Moscow, Russia. Institute of Molecular Genetics, Russian Academy of Sciences , Moscow, Russia.Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences , Moscow, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Peter the Great St. Petersburg Polytechnic University , Saint Petersburg, Russia.Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences , Moscow, Russia. Institute of Molecular Genetics, Russian Academy of Sciences , Moscow, Russia.Skolkovo Institute of Science and Technology, Center of Life Sciences , Moscow, Russia.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32564655

Citation

Selkova, Polina, et al. "Position of Deltaproteobacteria Cas12e Nuclease Cleavage Sites Depends On Spacer Length of Guide RNA." RNA Biology, 2020, pp. 1-8.
Selkova P, Vasileva A, Pobegalov G, et al. Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA. RNA Biol. 2020.
Selkova, P., Vasileva, A., Pobegalov, G., Musharova, O., Arseniev, A., Kazalov, M., Zyubko, T., Shcheglova, N., Artamonova, T., Khodorkovskii, M., Severinov, K., & Fedorova, I. (2020). Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA. RNA Biology, 1-8. https://doi.org/10.1080/15476286.2020.1777378
Selkova P, et al. Position of Deltaproteobacteria Cas12e Nuclease Cleavage Sites Depends On Spacer Length of Guide RNA. RNA Biol. 2020 Jun 21;1-8. PubMed PMID: 32564655.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA. AU - Selkova,Polina, AU - Vasileva,Aleksandra, AU - Pobegalov,Georgii, AU - Musharova,Olga, AU - Arseniev,Anatolii, AU - Kazalov,Maksim, AU - Zyubko,Tatyana, AU - Shcheglova,Nataliia, AU - Artamonova,Tatyana, AU - Khodorkovskii,Mikhail, AU - Severinov,Konstantin, AU - Fedorova,Iana, Y1 - 2020/06/21/ PY - 2020/6/23/entrez KW - AsCas12a KW - AsCpf1 KW - CRISPR-Cas KW - CasX KW - DpbCas12e KW - SpCas9 KW - cut site mapping KW - sgRNA SP - 1 EP - 8 JF - RNA biology JO - RNA Biol N2 - 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. SN - 1555-8584 UR - https://www.unboundmedicine.com/medline/citation/32564655/Position_of_Deltaproteobacteria_Cas12e_nuclease_cleavage_sites_depends_on_spacer_length_of_guide_RNA L2 - http://www.tandfonline.com/doi/full/10.1080/15476286.2020.1777378 DB - PRIME DP - Unbound Medicine ER -
Try the Free App:
Prime PubMed app for iOS iPhone iPad
Prime PubMed app for Android
Prime PubMed is provided
free to individuals by:
Unbound Medicine.