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Pré-Publication, Document De Travail Année : 2021

Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

Regina Tkach
  • Fonction : Auteur
Natalia Nikitchina
  • Fonction : Auteur
Nikita Shebanov
  • Fonction : Auteur
Vladimir Mekler
  • Fonction : Auteur
Egor Ulashchik
  • Fonction : Auteur
Olga Sharko
  • Fonction : Auteur
Vadim Shmanai
  • Fonction : Auteur
Konstantin Severinov
Nina Entelis
Ilya Mazunin
  • Fonction : Auteur
  • PersonId : 1116222

Résumé

ABSTRACT 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.
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Dates et versions

hal-03420212 , version 1 (09-11-2021)

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Regina Tkach, Natalia Nikitchina, Nikita Shebanov, Vladimir Mekler, Egor Ulashchik, et al.. Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA. 2021. ⟨hal-03420212⟩

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