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High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.
Nomura, Toshihisa; Kim, June-Silk; Ishikawa, Marumi; Suzuki, Kengo; Mochida, Keiichi.
Afiliación
  • Nomura T; RIKEN Center for Sustainable Resource Science, Yokohama, Japan.
  • Kim JS; RIKEN Baton Zone Program, Yokohama, Japan.
  • Ishikawa M; Faculty of Agriculture, Yamagata University, Tsuruoka, Japan.
  • Suzuki K; RIKEN Center for Sustainable Resource Science, Yokohama, Japan.
  • Mochida K; Institute of Plant Science and Resources, Okayama University, Okayama, Japan.
Microb Biotechnol ; 17(2): e14393, 2024 Feb.
Article en En | MEDLINE | ID: mdl-38332568
ABSTRACT
Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Euglena gracilis / Edición Génica Idioma: En Revista: Microb Biotechnol Año: 2024 Tipo del documento: Article País de afiliación: Japón

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Euglena gracilis / Edición Génica Idioma: En Revista: Microb Biotechnol Año: 2024 Tipo del documento: Article País de afiliación: Japón
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