High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.
Microb Biotechnol
; 17(2): e14393, 2024 Feb.
Article
in 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Euglena gracilis
/
Gene Editing
Language:
En
Journal:
Microb Biotechnol
Year:
2024
Document type:
Article
Affiliation country:
Japan
Country of publication:
United States