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

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