High‐efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis

Abstract

AbstractTransgene‐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.