CRISPR/LbCas12a‐mediated targeted mutation of Gracilariopsis lemaneiformis (Rhodophyta)

The CRISPR/Cas genome editing system has achieved high popularity in recent years (Knott and Doudna, 2018). It has already been used successfully in several plant species to perform gene knockout (Li et al., 2020), activation or repression (Li et al., 2019), and to target several sites simultaneously across the genome (Hu et al., 2019). In the present study, the CRISPR/LbCas12a (Lachnospiraceae bacterium ND2006) system was preliminarily established in Gracilariopsis lemaneiformis, an economically important red algae. Several base substitutions, as well as base insertions and deletions upstream of the target site, were detected. The study provides an important reference for the construction of macroalgae gene-editing systems. Six targets were initially selected on the carbonic anhydrase sequence (Figure S1a). DNA template for in vitro transcript was designed for each target (Figure S1b), synthesized by fill-in PCR using T7 primer and a unique guide RNA (gRNA) primer (Tables S1 and S2). Subsequently, each gRNA was transcribed in vitro (Figure S1c). The activity of LbCas12a was tested on the PCR amplified ca fragment (864 bp) combined with pre-incubated LbCas12a and gRNAs at 25 °C for 30 min (Table S1 CAF&R, Table S2). CAgRNA2 and CAgRNA3 displayed obvious cleavage bands (Figure 1a). Two-hundred tips of algae were bombardment-treated by ribonucleoprotein (RNP) complex containing CAgRNA2 and CAgRNA3 (Table S2). Five days later, ca gene of the tips was amplified using detection primers (Table S1 CAF&R, Table S2). The mutation sequences were enriched in the PCR products using the Single Strand Conformation Polymorphism (SSCP) technique (Figure 1b). Compared to the control group, weaker different bands were observed in the experimental group. The differential bands were recovered and then subjected to clonal sequencing. Compared to the wild-type sequence, two sequencing samples with base substitution were observed near the editing site (Figure 1d), one at 17 bases downstream of the Protospacer Adjacent Motif (PAM) site, and the other at 32 bases downstream of the PAM site, both of which were replaced by G from the original A (Figure 1e). To provide an easily observable trait, a pigment protein, the γ subunits of phycoerythrin (γpe) were also selected as a target gene. In the Gp. lemaneiformis (SRR20338037) genome, four sequences annotated as γpe were observed (Figures S3–S6). The motif compositions of the protein sequences were very similar; however, the similarity between DNA sequences was low (Figure S1d). For each sequence of γpe, two sites were selected, respectively (Figures S3–S6), and the possibility of off-target effects was reduced through genome alignment. Similarly, the DNA templates for pre-CRISPR RNA (pre-crRNA) were generated by fill-in PCR and transcribed to obtain different gRNAs (Tables S1 and S2). LbCas12a activity was also tested in vitro at 25 °C for 30 min (Table S1 PE1F&R, PE2F&R, PE3F&R, PE4F&R, Table S2). Cleavage could be observed in the second gRNA of pe2 (PE2gRNA2). No obvious cleavage was observed in other experimental groups (Figure 1f). PE2gRNA2 was selected for further activity tests. The LbCas12a activity was then tested at 37°C to ensure that gRNA was effective (Figure 1c). Cleavages could be clearly observed for PE2gRNA2 at 37°C and the products were of the expected sizes (364 and 470 bp). After microparticle bombardment, the algae were cultured at 25 °C for 2 day, and then cultured at 20 °C. Fifteen days later, the algal tips were checked under fluorescence microscopy. Different spots were observed on the surfaces of the branches. In some areas, the spots were scattered and dotted, while in other areas, patches were formed (Figure 1g). Under different filters, the patches were distinct from the surroundings, with blue, dark, and bright green patches observed under DAPI, TRITC and FITC filters respectively. The patches were collected using scalpels. Pe was amplified by PCR (Table S1 PE2F&R). The sequencing results (Figure 1h,i) showed that near the editing site, base substitution existed in two clones, namely, in the #21 clone, the 67th base upstream of the PAM site gene was shifted from T to C. In the #63 clone, the base substitution occurred at the third base of the PAM site, which shifted from A to G. In addition, other sequence mutations occurred in the vicinity of 200 bases upstream of the PAM site (Figure S1e). Among them (Figure 1j,k), the 145th base T deletion occurred in the #72 clone. In the #98 clone, base C was inserted after the 142nd base, and the 144th base shifted to G from T. In the #102 clone, base C was inserted after the 142nd base. In the #113 clone, base C was inserted after the 76th base. In the #115 clone, base C was inserted after the 76th base, and 144th base shifted to G from T. The insertion or deletion of single bases in the mutant sequences above led to shifts in the open reading frame, which completely altered the amino acid sequences. This study, for the first time, established a gene-editing system for Gp. lemaneiformis. Using microparticle bombardment to directly transform RNP into algal tips greatly simplifies the experimental procedures when compared with conventional plasmid systems, and the SSCP method was confirmed to facilitate the screening of editing results in numerous wild-type cells. To identify gRNAs with high efficiency at 25 °C, more than three gRNA options are required. For macroalgae, this study confirmed for the first time that gene editing could be achieved by the CRISPR/LbCas12a system, and the selection of genes with suitable phenotypes could facilitate the screening of editing results. Our study lays a foundation for gene editing work in Gp. lemaneiformis and other macroalgae, and offers key insights. This work was supported by the China Agriculture Research System of MOF and MARA (CARS-50), National Natural Science Foundation of China (NO. 32072953). The authors declare no conflict of interest. ZHS, YYH and ME designed the experiments. JYZ, QW, RC, NZ and HHC performed the experiments. ME provided technical supports. JYZ, QW and ME wrote the manuscript. ZHS, YYH and ME supervised the research. All the authors read and approved the manuscript. Appendix S1 Materials and methods. Table S1 List of primers. Table S2 Reaction systems. Figure S1–S6 Target gene sequence and target position. 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