High‐throughput sequencing revealed low‐efficacy genome editing using Cas9 RNPs electroporation and single‐celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis

• DOI: 10.1111/pbi.13915
• Publication Date: 2022-08-29T08:49:50Z
• Authors: Zhenfan Chen; Jiayi Zhu; Zixi Chen; Ming Du; Rao Yao; Wen Fu; Anping Lei; Jiangxin Wang
• Keywords: Euglena gracilis; Guide RNA; Subgenomic mRNA

The genus Euglena contains more than 1000 species of single-celled flagellated microorganisms with both plant and animal characteristics. As a model organism, E. gracilis has been studied well to address fundamental questions in chloroplast development photosynthesis, implications physiology, biochemistry cell biology (Schwartzbach Shigeoka, 2017). However, research bioengineering biotechnology Euglena, especially using genome editing, remains insufficient (Figure 1a). Nomura et al. (2019) reported successful CRISPR/Cas9-mediated editing electroporation Cas9 ribonucleoproteins (RNPs) targeting EgGSL2 obtained mutant rates approximately 70–90% based on morphology amplicon-sequencing (Nomura 2019). protocol for implementing the CRISPR experiments was also described further detail published al., 2020). To date, these are only articles related gracilis. Since their publication, no other case or report by groups, including ours. In this study, we repeated 2019, 2020) assessed efficiency high-throughput sequencing. Further, sgRNA crtP1 gene phytoene desaturase (PDS) designed as reference (Method S1). EG300 experimental procedures parameters strictly followed (2020) 1c). Moreover, protoplast-like cells (Plas) treated proteinase K were used extended voltage from 150 600 V two types devices, NEPA21 Bio-Rad Xcell Additional details process Method S1. result showed that CRISPR-RNPs system worked efficiently induce enzymatic cleavage partial vitro 1b). T7 Endonuclease I (T7E I) assays three replications indicated low 'target1' 1d). sequencing amplicons performed Illumina NovaSeq 6000 platform. After data control, sequences aligned operational taxonomic units (OTUs) clustered (FLASH v1.2.11 USEARCH v10.0.240); OTUs found be most high-quality sequences, which represented wild type, mutant1 mutant2 1e) (accessible set China National GeneBank (CNGB); accession no.: CNP0002995). available ranging 29,541 353,307 eight samples. one sample, EG450, revealed (0.81%) (0.17%), respectively 1f). results but had very efficiency. samples except EG450 0% With gene, an rate 3% total CNGB; CNP0003142) These indicate delivery RNPs into is difficult. Microinjection physical method deliver small volume substances at appropriate location, such cytoplasm nucleus (Zhang Yu, 2008). It visible real-time traceable widely zebrafish embryos mouse zygotes because its high lethality (Gordon 1980; Yuan Sun, 2009). application microinjection microalgae, although reported, rare (Nichols Rikmenspoel, 1978), if size less 100 μm. Here, present exogenous materials cells. For example, dihydrochloride (DAPI) stain successfully injected TransferMan 4r FemtoJet 4i (Eppendorf, Germany), blue fluorescence observed nuclear region excitation/emission wavelengths (364 nm/454 nm) under DMi3000B epifluorescence microscope (Leica, Germany) 1g). gracilis, PDS target1 used. group, six among survived, each clone sequenced verify specific mutation. According DNA results, precise gene. cytosine base deleted 295 sequence colour differences between WT crtP1-mutant evident same density 2.4 × 106 cells/ml 1h). 16.7% surviving cells, whereas 1.0% calculated number processed technology coupled maintained even after cultivation year. could though twelve clones survived. not newly emerging technique rather advanced sophisticated technique, primarily when smaller 20 μm (Chen 2022). presents sphere-shaped flexible pellicle, hinders manipulation microinjection. open tip injection pipette should 500 nm, tips 50 nm suitable reduce mortality. pressure varies 2500 hPa depending circumstances. Several industrial microalgae tiny thick walls movability. This may explain why compared mammalian rare. study represents first delivering CRISPR/Cas9 microalgal summary, experiment (2020), suboptimal maximum 0.98% electroporation. conducted knocked out relatively (16.7%). generated stable can good candidate studying carotenoid metabolism best our knowledge, microalgae. Overall, demonstrate microinjection-based potential material facilitate We thank Instrument Analysis Center Shenzhen University. work partially supported China's Key R&D Programs (2018YFA0902500; 2020YFA0908703; 2021YFA0910800) Natural Science Foundation (41876188). authors declare conflicts interest. ZC JW conceived experiments. JZ, ZC, MD, RY WF helped perform AL revise manuscript. All read approved final Figure S1 Mutant targeted electroporation, determined Methods Movie Short movie Please note: publisher responsible content functionality any supporting information supplied authors. Any queries (other missing content) directed corresponding author article.