Non-motile photosynthetic flagellates sediment well and thus can be harvested more easily under mass cultivation (Brennan and Owende, 2010). However, a precise genetic manipulation of microalgal motility remains challenging. The nutrient-rich microalga Euglena gracilis is widely used in the food, cosmetics and feed industries (Suzuki, 2017). This alga accumulates paramylon, a crystalline β-1,3-glucan with multiple industrial applications (Harada et al., 2020). Under hypoxia, E. gracilis degrades paramylon to generate energy and converts it into wax esters, primarily myristic acid (C14:0) and myristyl alcohol (C14:0) (Inui et al., 2017), which are a potential source of jet biofuel. To improve the harvesting efficiency of E. gracilis, a non-motile mutant (${\mathrm{M}}_3^{-}\text{ZFeL}$) was generated using Fe-ion beam irradiation. However, ${\mathrm{M}}_3^{-}\text{ZFeL}$ grew slower and produced less lipids than the wild type (WT), suggesting that the mutagenesis caused undesirable side mutations (Muramatsu et al., 2020). Although flagellar mutants with motility defects have been identified in model organisms such as Chlamydomonas reinhardtii, Trypanosoma brucei and Caenorhabditis elegans (Kamiya et al., 1991; Wingfield et al., 2018), a precise manipulation of motility in E. gracilis remains to be explored, despite its industrial usefulness.

Here, using our Cas9 ribonucleoprotein (RNP)-based genome-editing technique (Nomura et al., 2019), we targeted the Bardet–Biedl syndrome (BBS) genes in E. gracilis to generate non-motile strains. We identified the genes encoding proteins homologous to Caenorhabditis elegans BBS-7 (NP_499585.1; tblastn E value 7e–72) or BBS-8 (NP_504711.2; tblastn E value 7e–123). These BBSome components are associated with the intraflagellar transport of particles and mediate the trafficking of cilium/flagellum membrane proteins in eukaryotic cells (Hammond et al., 2021; Nakayama and Katoh, 2018; Wingfield et al., 2018). We designed two pairs of single guide RNAs (sgRNAs) that target different regions of EgBBS7 and EgBBS8 and introduced Cas9–sgRNA RNPs into E. gracilis cells by electroporation (Method S1, Table S1).

Assessing the motility of the electroporated E. gracilis cells, we identified four sets of target sequences and protospacer adjacent motifs (PAMs), which are useful for inducing the stable non-motility phenotype in E. gracilis (EgBBS7-A and EgBBS7-B; and EgBBS8-B and EgBBS8-D). To establish clonal bbs strains, we isolated two cell lines each for bbs7 and bbs8 (Method S1). PCR-based genotyping detected 500–1500 bp deletions within the EgBBS7 and EgBBS8 target regions in these strains (Method S2, Figure 1a,b), and Sanger sequencing verified that large genomic regions were deleted, including intronic regions (Figure 1c,d). When isolated single cells were cultured, all eight bbs mutant strains flocculated, whereas the WT spread (Figure 1e). We confirmed the non-flagellar phenotype of the mutant strains by scanning electron microscopy (Method S3, Figure 1f,g) and their non-motility by a trace momentum assay that quantifies swimming motion (Method S4, Figure 1h,i). The results indicate that EgBBS7 and EgBBS8 contribute to forming a full-length flagellum in E. gracilis and are thus required for motility.

Next, we assessed gravitational sedimentation of the non-motile bbs mutants by time-lapse imaging in 1-min intervals (Figure 1j). We created 2D kymographs representing time-dependent changes in the transparent–sediment interface level in bbs and WT cells, revealing the rapid sedimentation of the bbs mutants (Method S5, Figure 1k). We examined time-series changes in the level of the transparent–sediment area in flasks segmented from binarized images. The WT cultures remained cloudy at 120 min after the flasks settled due to the presence of swimming cells, whereas bbs cells almost fully sedimented (Method S5, Figure 1l). Moreover, comparing the dry weights of bbs and WT sediments at 100 min after the flasks settled showed that the bbs mutants had 32–38% higher sedimentation rates than WT (Method S5, Figure 1m).

We also examined the growth, biomass, paramylon content and lipid content of WT and the bbs mutants (Method S6). Using the cell density of cells cultured in KH medium as estimate for growth, the growth rates of all four bbs strains were not significantly different from WT until 4 days after culture initiation, but the cell concentration of the bbs8 mutants was slightly higher than that of WT (p^bbs8-B #4 = 0.042, p^bbs8-D #13 = 0.011, Dunnett's test) at 7 days after culture initiation (Figure 1n). We did not detect marked differences between the bbs mutants and WT in terms of biomass harvested at 7 days after culture initiation or for the lipid content of cells under aerobic or hypoxic conditions (Figure 1o,p). Interestingly, the paramylon content of cells under aerobic conditions was significantly higher in the bbs mutants than in the WT (Figure 1q), which might be related to cellular motility and paramylon biosynthesis and/or accumulation. These results suggest that mutations in the EgBBS genes that facilitate sedimentation do not negatively affect the production of biomass or high-value products.

During the mass cultivation of microalgae, harvesting accounts for 20–30% of total production costs; therefore, improving harvesting procedures will enhance the economic viability of microalgal biomass production (Brennan and Owende, 2010). We estimated the harvesting efficiency of the bbs mutants to be 32–38% higher than that of the WT (Figure 1m), while all strains produced similar amounts of biomass (Figure 1o, aerobic). Therefore, our results demonstrate that knocking out EgBBS genes in E. gracilis could improve harvesting efficiency without negatively affecting productivity.

非运动的光合鞭毛可以很好地沉积，因此在大规模栽培下更容易收获（Brennan 和 Owende， 2010 ）。然而，微藻运动的精确遗传操作仍然具有挑战性。营养丰富的微藻Euglena gracilis广泛应用于食品、化妆品和饲料行业（Suzuki， 2017 ）。这种藻类会积累裸藻淀粉，这是一种具有多种工业应用的结晶 β-1,3-葡聚糖（Harada等人， 2020 ）。在缺氧条件下， E. gracilis降解裸藻淀粉以产生能量，并将其转化为蜡酯，主要是肉豆蔻酸 (C14:0) 和肉豆蔻醇 (C14:0)（Inui等人， 2017 ），它们是喷射的潜在来源生物燃料。为了提高E. gracilis （一种非运动突变体）的收获效率（ ${\mathrm{M}}_3^{-}\text{ZFeL}$ ）是使用铁离子束照射产生的。然而， ${\mathrm{M}}_3^{-}\text{ZFeL}$ 与野生型 (WT) 相比，它们生长更慢，产生的脂质更少，这表明诱变导致了不良的副突变 (Muramatsu et al ., 2020 )。尽管在莱茵衣藻、布氏锥虫和秀丽隐杆线虫等模型生物中发现了具有运动缺陷的鞭毛突变体（Kamiya等， 1991 ；Wingfield等， 2018 ），但对E. gracilis运动的精确控制仍有待进一步研究。尽管它具有工业用途，但仍被探索过。

在这里，我们使用基于 Cas9 核糖核蛋白 (RNP) 的基因组编辑技术 (Nomura et al ., 2019 )，我们针对E. gracilis中的Bardet-Biedl 综合征 (BBS)基因来生成非运动菌株。我们鉴定了编码与秀丽隐杆线虫BBS-7 (NP_499585.1; tblastn E值 7e–72) 或 BBS-8 (NP_504711.2; tblastn E值 7e–123) 同源的蛋白质的基因。这些 BBSome 成分与颗粒的鞭毛内运输相关，并介导真核细胞中纤毛/鞭毛膜蛋白的运输（Hammond等， 2021 ；Nakayama 和 Katoh， 2018 ；Wingfield等， 2018 ）。我们设计了两对针对EgBBS7和EgBBS8不同区域的单引导 RNA (sgRNA)，并通过电穿孔将 Cas9–sgRNA RNP 引入E. gracilis细胞中（方法 S1，表 S1）。

通过评估电穿孔E. gracilis细胞的运动性，我们鉴定了四组靶序列和原型间隔子相邻基序 (PAM)，它们可用于诱导E. gracilis中稳定的非运动表型（ EgBBS7 -A 和EgBBS7 -B；和EgBBS8 -B和EgBBS8 -D)。为了建立克隆bbs菌株，我们分离了bbs7和bbs8的两个细胞系（方法 S1）。基于 PCR 的基因分型检测到这些菌株中EgBBS7和EgBBS8目标区域内有 500–1500 bp 缺失（方法 S2，图 1a、b），Sanger 测序验证了大基因组区域被删除，包括内含子区域（图 1c、d） 。当培养分离的单细胞时，所有八个bbs突变株均絮凝，而 WT 则扩散（图 1e）。我们通过扫描电子显微镜（方法 S3，图 1f，g）确认了突变菌株的非鞭毛表型，并通过量化游泳运动的微量动量测定（方法 S4，图 1h，i）确认了它们的非运动性。结果表明， EgBBS7和EgBBS8有助于在E. gracilis中形成全长鞭毛，因此是运动所必需的。

接下来，我们通过 1 分钟间隔的延时成像评估了非运动bbs突变体的重力沉降（图 1j）。我们创建了 2D kymographs，代表bbs和 WT 细胞中透明沉积物界面水平随时间的变化，揭示了bbs突变体的快速沉降（方法 S5，图 1k）。我们检查了从二值化图像中分割出来的烧瓶中透明沉积物区域水平的时间序列变化。由于存在游动细胞，WT 培养物在烧瓶沉降后 120 分钟仍保持浑浊，而bbs细胞几乎完全沉降（方法 S5，图 1l）。此外，比较烧瓶沉降后 100 分钟时bbs和 WT 沉积物的干重表明， bbs突变体的沉降速率比 WT 高 32-38%（方法 S5，图 1m）。

我们还检查了 WT 和bbs突变体的生长、生物量、裸藻淀粉含量和脂质含量（方法 S6）。使用在 KH 培养基中培养的细胞密度作为生长估计，直到培养开始后 4 天，所有四种bbs菌株的生长速率与 WT 没有显着差异，但 bbs8 突变体的细胞浓度略高于 bbs8 突变体的细胞浓度。培养开始后 7 天时的 WT（p ^bbs8-B #4 = 0.042，p ^bbs8-D #13 = 0.011，Dunnett 检验）（图 1n）。我们没有检测到bbs突变体和 WT 在培养开始后 7 天收获的生物量或有氧或缺氧条件下细胞的脂质含量方面存在显着差异（图 1o，p）。有趣的是，有氧条件下bbs突变体细胞的裸藻淀粉含量显着高于WT（图1q），这可能与细胞运动和裸藻淀粉生物合成和/或积累有关。这些结果表明，促进沉淀的EgBBS基因突变不会对生物质或高价值产品的生产产生负面影响。

在微藻大规模养殖过程中，收获成本占总生产成本的20-30%；因此，改进收获程序将提高微藻生物质生产的经济可行性（Brennan 和 Owende， 2010 ）。我们估计bbs突变体的收获效率比 WT 高 32-38%（图 1m），而所有菌株产生的生物量相似（图 1o，需氧）。因此，我们的结果表明，敲除E. gracilis中的EgBBS基因可以提高收获效率，而不会对生产力产生负面影响。