Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA
| Autor: | Nina Entelis, Konstantin Severinov, Vadim V. Shmanai, Natalia Nikitchina, Ilya O. Mazunin, Nikita Shebanov, Regina Tkach, Vladimir Mekler, Egor A. Ulashchik, Ivan Tarassov, O. L. Sharko |
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| Přispěvatelé: | Génétique moléculaire, génomique, microbiologie (GMGM), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2020 |
| Předmět: |
Trans-activating crRNA
0303 health sciences Nuclease biology Effector [SDV]Life Sciences [q-bio] RNA Cleavage (embryo) Cell biology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine chemistry Genome editing biology.protein CRISPR 030217 neurology & neurosurgery DNA 030304 developmental biology |
| DOI: | 10.1101/2020.12.21.423781 |
| Popis: | CRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases. |
| Databáze: | OpenAIRE |
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