Gene editing of economic macroalga <i>Neopyropia yezoensis</i> (Rhodophyta) will promote its development into a model species of marine algae

Abstract

Macroalga aquaculture in China dates back to 960–1279 AD and currently represents an impressive 60% of global production (Blouin et al., 2011; FAO, 2022). Among the various aquacultural algae, Neopyropia yezoensis holds the highest economic value per unit, with an annual production exceeding 2.2 million tons (FAO, 2022). Presently, the predominant breeding techniques for N. yezoensis include natural seed selection and artificial mutation breeding. However, these methods are characterized by inefficiency and inadequacy for the development of high-quality varieties. Furthermore, the reproductive characteristics of N. yezoensis, marked by hermaphroditism and self-fertilization (Blouin et al., 2011; He et al., 2019), pose formidable challenges to hybrid breeding. Therefore, there is a pressing need for improved breeding strategies for N. yezoensis. The intricate life cycle of N. yezoensis, featuring a haploid gametophytic macro-stage and a diploid sporophytic micro-stage (Blouin et al., 2011; He et al., 2019), sets it apart from terrestrial organisms, highlighting its unique position in biological evolution. Neopyropia yezoensis is not only an economically important species; its unique properties give it the potential to be a model organism. For example, it can complete its life cycle in the laboratory, has fewer chromosomes, a compact genome, and exhibits rapid growth (He et al., 2019; Wang et al., 2020). Despite its importance, the potential of N. yezoensis as a model species has been hindered by the stagnation in genetic manipulation methods, which are crucial for advancing reverse genetics research. CRISPR/Cas are targeted cleavage systems, in which the Cas nuclease cleaves DNA sequences flanking a protospacer adjacent motif (PAM) under gRNA guidance (Jinek et al., 2012). The CRISPR/Cas gene editing systems have been a cornerstone of biotechnology research for more than a decade, and its scope has been expanded to include variants such as CRISPR/Cas9, CRISPR/Cas12a (formerly Cpf1) and CRISPR/Cas13a (formerly C2c2) (Murugan et al., 2017). These tools facilitate precise genome editing across a diverse array of cell types and organisms (Knott & Doudna, 2018), including some marine species. Recent studies have successfully applied CRISPR/Cas9 and CRISPR/LbCas12a for targeted mutagenesis in the model brown alga Ectocarpus sp7 and red alga Gracilariopsis lemaneiformis using ribonucleoprotein (RNP) delivery (Badis et al., 2021; Zhang et al., 2023), underscoring the broadening potential of these gene editing tools. However, efficiently producing gene knockout mutants of red macroalgae remains significant challenges. This study pioneers the development of an efficient gene editing system in N. yezoensis, a species characterized by a high-genomic GC content and issues related to the silencing of exogenous gene expression (Shin et al., 2016; Wang et al., 2020). For the first time, we successfully edited a target gene using CRISPR/Cas9 plasmid system in N. yezoensis, produced a plethora of gene editing types, and obtained a small number of homozygous knockout mutants. This groundbreaking achievement not only lays a solid foundation for the molecular breeding in N. yezoensis but also propels it forward as a potential model species for marine algae studies. Our methodology involved cloning the N. yezoensis ubiquitin gene promoter PypUbi1 for expressing the GUS reporter gene, determining the optional length of the PypUbi1 promoter (Fig. 1a; Supporting Information Table S1), and constructing the expression cassette PypUbi1::Cas9-P2A-Hyg-NosT (Fig. 1c). Previous studies have demonstrated the expression of codon-optimized GUS and hygromycin resistance gene (Hyg) in N. yezoensis (Fukuda et al., 2008; Uji et al., 2014). Therefore, this study employed the GUS reporter gene for identifying endogenous promoters in N. yezoensis and used the Hyg as a selection marker to obtain resistant plants (Table S2). The use of the self-cleaving peptide P2A (Kim et al., 2011), validated for functionality in N. yezoensis (Fig. S1a,b), played a pivotal role in facilitating simultaneous Cas9 expression and hygromycin resistance enrichment screening. Given that gRNA expression is typically driven by a polymerase III (pol III) promoter (Cong et al., 2013), the identification of a suitable pol III promoter in N. yezoensis was a critical step. Using the cRT-PCR technique (Hang et al., 2015; Methods S1), identification (Figs 1b, S1c,d) and utilization of the PyU6 promoter for gRNA expression established the molecular foundation for the working of the CRISPR/Cas9 editing system in N. yezoensis. Notably, the U6 promoter was used as a pol III promoter for gRNA expression, demonstrating certain pol II promoter activity (Gao et al., 2018; Fig. 1b). To demonstrate the applicability of this system, a serine/threonine kinase gene (Py02079) in N. yezoensis was subjected to testing, and the gRNA sequence was designed according to Concordet & Haeussler (2018) (Fig. S1e; Table S1; Methods S1). Using the CRISPOR (Concordet & Haeussler, 2018) website and the genome ASM982973v1 as a reference, we selected a crRNA with the highest score in the exons near the 5′ end of the target gene. In vitro validation confirmed that the selected gRNA exhibited a high-cleavage efficiency (Fig. S1f–h). A vector pV5Py62 consisting of the Cas9 expression cassette and gRNA (crRNA-tracrRNA) expression cassette was constructed (Fig. 1c; Methods S1), and delivered into the thalli of N. yezoensis via biolistic transformation. Following hygromycin screening, 24 resistant algae seedlings were subjected to DNA-level validation. Results confirmed the successful integration of Cas9 and gRNA expression cassettes into the genomes of these 24 algae strains (Fig. 1d,e). Notably, amplification of the target site revealed that strain 18 had higher molecular weight than expected (Fig. 1f). Sanger sequencing of the PCR amplicon revealed a 6 bp deletion upstream of the PAM site in strain 18, accompanied by a 414 bp fragment insertion (Fig. 2a). This caused premature translation termination, truncating the protein from 681 amino acids to 125 amino acids, thus resulting in gene knockout. Alignment of the 414 bp sequence indicated its origin from the N. yezoensis genome (Table S3), likely due to relatively rare recombination or insertion of large fragments during nonhomologous end joining. Further analysis of sequencing peak graphs for the other 23 individual strains showed overlapping peaks near the PAM site, such as thalli 1, 2, 3, 5, 6, 7, 8, 9, 10, 19, 21, and 22 (Fig. 2b), suggesting heterozygous properties of the target gene in each strain. TIDE (Brinkman et al., 2014) analysis of Sanger sequencing data from chimeras 3, 5, 8, and 22 revealed indel frequencies of 43.4%, 29.7%, 28.8%, and 25.9%, respectively (Fig. S2). T-vector cloning results revealed indel ratios of 70%, 40%, 60%, and 40% for the four chimeras, respectively (Fig. S3). It is noteworthy that the thallus of N. yezoensis is in a haploid gametophytic stage, thus the different alleles of the target gene observed in each strain may be due to different indel in each cell of the thalli, or gene silencing in some cells, resulting in the differential expression of Cas9 or gRNA. Real-time quantitative polymerase chain reaction experiments were performed on five strains (3, 5, 8, 18, and 22) to examine the expression of Cas9 and gRNA (Methods S1). Results revealed substantial Cas9 and gRNA expression levels in the homozygote, exceeding those of the housekeeping gene PyAct1. However, while Cas9 and gRNA were expressed in chimeric strains, their expression levels were significantly lower than the homozygous strain (Table S4). The findings indicated that, while exogenous gene expression was achievable in N. yezoensis, a significant portion of individuals exhibited reduced expression levels. The phenomenon of gene silencing could partially explain the generation of chimeras. Although transgene silencing (Shin et al., 2016) and co-suppression (Zheng et al., 2021) in N. yezoensis has been reported previously, the mechanisms underlying this phenomenon require further investigation. One month after the initial screening of 24 individuals, we continued mutant screening and sequenced a second batch of 49 strains. These individuals originated from the same initial experiments. The sequencing results confirmed six homozygous mutations (Fig. S4). In total, the proportion of homozygous mutations was 9.6% (7/73). Although this ratio is not outstanding, this represents a promising advancement. To further characterize the gene editing system in N. yezoensis, we conducted experiments to obtain more watertight data. DNA was extracted from a mixture of 150 seedlings isolated from the total batch of resistance screening and was used as template for amplification of the target site with the Py02079 F/R primer (Table S1). The PCR products were subjected to high-throughput SNP sequencing with an average sequencing depth exceeding 50 000× (Methods S1). The results revealed numerous SNPs near the target site (Dataset S1), with gene edits primarily distributed within 6 bp upstream of the PAM site. The most frequent mutation site was the −3 position upstream of the PAM, with a SNP ratio of 37.43%, followed by 36.94% (−2), 34.59% (−4), 29.19% (−1), 28.52% (−5) and 27.97% (−6), respectively (Fig. 2c,d). These high-frequency sites were consistent with the typical characteristics of the CRISPR/Cas9 gene editing system (Jinek et al., 2012). The remarkable breakthrough reported in this work is underscored by the identification and successful utilization of the endogenous PyU6 promoter and the incorporation of the self-cleaving peptide P2A. The use of P2A as a connector between Cas9 and Hyg significantly improved the efficiency of resistance enrichment screening, facilitating the success of gene editing in N. yezoensis. For the first time, instead of using RNP delivery, a screenable CRISPR/Cas9 gene editing system at the molecular level signifies a paradigm shift in macroalgal gene editing methodologies. In conclusion, this study lays the foundation for genome editing technology using CRISPR/Cas9 in N. yezoensis. This progress represents a pivotal step toward advancing precision design breeding in this economically significant macroalga and provides essential reverse genetics tools for the study of red algae biology. The trajectory set by this study foresees a new era in the molecular understanding of N. yezoensis and anticipates a revolutionary impact on the economic seaweed industry. We would like to express our great appreciation to Dr Per Winge from the Department of Biology, Norwegian University of Science and Technology for his valuable advice on identifying the U6 gene of N. yezoensis. We thank Dr Satoru Fukuda and Toshiki Uji from the Faculty of Fisheries Sciences, Hokkaido University, for generously providing the codon-optimized GUS and hygromycin resistance gene. We are grateful to Dr Wenjun Wang from Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, for her assistance in revising the manuscript. This work was supported by the Major Scientific and Technological Innovation Project of Shandong Provincial Key Research and Development Program (2022LZGC004), the National Key R&D Program of China (2018YFD0901500), the Research Fund for the Taishan Scholar Project of Shandong Province (tspd20210316), the National Natural Science Foundation of China (42376091, 41876160), and the Opening Foundation of Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, CAS (2023FB12). None declared. GW, XX and HW conceived and designed this research. HW and XX performed the experiments, analyzed the data and wrote the manuscript. GW and WG revised the manuscript. ZZ and JZ assisted in the biolistic bombardment. ZS and LH assisted in performing cRT-PCR experiments. BZ, JN, SG and XW gave advice during the data analysis. HW and XX contributed equally to this work. More detailed data are available in the Supporting Information. Supplementary figures related to the study can be found in Figs S1–S4. For sequence information required for experimental integrity, refer to Tables S1–S3. Real-time quantitative polymerase chain reaction results are presented in Table S4. Materials and methods used in this study are available in Methods S1. SNPs sequencing results from targeted gene editing are available in Dataset S1. Dataset S1 All the SNPs. Fig. S1 Functional analysis of P2A and schematic diagram of the PyU6 gene and in vitro cleavage of the target sequence. Fig. S2 TIDE analysis of indel frequencies in chimeric strains 3, 5, 8, and 22. Fig. S3 Monoclonal decomposition of PCR products of chimeric strains 3, 5, 8 and 22. Fig. S4 Peak map of six homozygous strains 30, 35, 50, 52, 58 and 64. Methods S1 Materials and Methods. Table S1 Primers used in this study. Table S2 Sequences of codon-optimized PyCas9, GUS and hygromycin resistance genes. Table S3 Sequence of the inserted 414 bp fragment of strain 18. Table S4 Results of real-time quantitative polymerase chain reaction. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. 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.

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