Construction of all-in-one PtTALEN-ARS plasmids and introduction of mutations in N. oceanica
To construct transgene-free genome edited N. oceanica, the vectors used for expressing genome-editing tools must be removed. The all-in-one PtTALEN vectors containing CEN/ARS were expected to behave as episomes in N. oceanica cells (Fig. 1A). Under non-selective conditions, the all-in-one PtTALEN vectors containing CEN/ARS may be removed spontaneously from host N. oceanica cells, since the vectors are not necessary for host cell growth.
Construction of all-in-one PtTALEN-ARS plasmids and verification of genome editing activity. (A) Workflow of transgene-free genome editing using all-in-one PtTALEN-ARS plasmids. The plasmids containing CEN/ARS are not inserted into the chromosome and can be removed after mutagenesis. Chr: chromosome. (B) Schematic diagram of all-in-one PtTALEN-ARS plasmids. These plasmids have two antibiotic genes, AmpR and KanR. These are facilitated to construct the all-in-one TALEN-ARS vectors. ProLHC: LHC promoter; terFCP: FCP terminator; L-TALEN: left TALEN; R-TALEN: right TALEN; AmpR: ampicillin resistance gene; ShBle: zeocin resistance gene; KanR: kanamycin resistance gene; ARS: yeast centromere and autonomous replication sequence. (C) Heteroduplex mobility assay (HMA) and Cel-I assay for TALEN target sites of NoNR genes in wild-type and TALEN-induced N. oceanica by the introduction of all-in-one PtTALEN-ARS or TALEN plasmids targeting NoNR. HMA was performed by amplifying the NoNR target site. The Cel-I assay was performed by treating NoNR PCR products with Cel-I enzymes. WT: Wild-type genomic DNAs; ARS-T: total DNAs from all emerged N. oceanica colonies following introduction of all-in-one NoNR PtTALEN-ARS plasmids; T: total DNAs from all emerged N. oceanica colonies following the introduction of all-in-one NoNR PtTALEN plasmids; M: DNA ladder marker.
Previously, we reported that the all-in-one PtTALEN plasmids possessed high mutagenesis activity; however, the insertion of the expression cassette of the right-TALEN into the left-TALEN expression plasmids was inefficient. This might be due to the large vector size. To improve the ease of construction of all-in-one PtTALEN plasmids, we initially aimed to use two antibiotic markers. The kanamycin resistance gene was inserted into the flanking region of the right-TALEN expression cassette. The resultant pMD20-TALEN47-KanR plasmid contained only the kanamycin resistance gene as an antibiotic marker. The integrated all-in-one plasmids possess both ampicillin and kanamycin resistance (Supplementary Fig. S1). Therefore, only strains harboring the newly integrated all-in-one plasmids are capable of growing on LB solid plates containing both ampicillin and kanamycin. In practice, colony PCR and restriction enzyme verification showed that all tested colonies harbored the expected all-in-one PtTALEN vectors (Supplementary Fig. S2). Next, we constructed all-in-one PtTALEN-ARS plasmids as shown in Fig. 1B. CEN/ARS was amplified using a yeast low-copy plasmid as a template and then inserted into the left-TALEN expression plasmids. DNA-binding modules for the endogenous nitrate reductase gene NoNR were as previously described17. Due to concern about the lowering mutagenesis activity by inserting ARS into all-in-one TALEN vectors, we attempted to confirm the genome editing activity of TALENs expressed from all-in-one PtTALEN-ARS plasmids targeting NoNR. All-in-one PtTALEN-ARS vectors were introduced into N. oceanica cells. All colonies on selective plates were collected, mixed, and subjected to extraction of total DNAs, which potentially contain both genomic and plasmid DNAs. In addition, the region around the NoNR target site (516 bp) was amplified using total DNAs. The genome editing activity of the all-in-one PtTALEN-ARS plasmid was assayed using the heteroduplex mobility assay (HMA) and Cel-I assay of the PCR products. The high mutagenesis activity of all-in-one PtTALEN plasmid without ARS was previously reported17. Therefore, total DNAs extracted from the colonies treated with all-in-one PtTALEN plasmid lacking ARS was used as a positive control. PCR products derived from all-in-one PtTALEN-ARS plasmid exhibited a band shift in the HMA that was similar to the positive control, suggesting the introduction of mutations (Fig. 1C). Moreover, the Cel-I assay detected cleavage bands for both all-in-one PtTALEN-ARS plasmids and the positive control (Fig. 1C). These results indicated that PtTALENs expressed from all-in-one PtTALEN-ARS plasmids exhibit genome editing activity in N. oceanica.
Clearance of all-in-one PtTALEN-ARS plasmids from N. oceanica cells
We attempted to remove the all-in-one PtTALEN-ARS plasmids from transformed cells. Transformants of all-in-one PtTALEN-ARS plasmids targeting NoNR were collected from selective plates. The NoNR target site of these transformants was confirmed by sequencing. Two NoNR mutants (Strain ID: AZ and BZ) were used for the following experiments. To remove the vectors, these strains were cultured using liquid or solid medium in the absence of zeocin, as shown in Fig. 2A. The 12 colonies that appeared on the solid medium without zeocin were collected, and the clearance of all-in-one PtTALEN-ARS plasmids was confirmed by PCR (Fig. 2B). A primer pair for amplification of the FokI nuclease domain sequence of TALEN was used. In all tested colonies, the intensity of FokI PCR bands was substantially reduced. In particular, six strains derived from AZ or BZ did not have detectable PCR bands for FokI (Strain ID: A5, A7, and A11 and Strain ID: B1, B2, and B3). The strains with potential plasmid removal were investigated again with three additional primer pairs for plasmid detection (Fig. 2C). The absence of the all-in-one PtTALEN-ARS plasmid was further verified by the absence of PCR bands for FokI, KanR and ZeoR in these strains. This result suggested that these strains do not harbor all-in-one PtTALEN-ARS plasmids. Subsequently, we checked the sequence of the NoNR target site to rule out possible wild-type strain contamination (Fig. 3A). Sequence analysis showed that the NoNR target site in these strains (Fig. 3A, Strain ID: A5, A7, A11, B1–B3) corresponded with that of the parental strains (Fig. 3A, Strain ID: AZ, BZ). Furthermore, we performed spot test using each type of solid plate for phenotypic validation (Fig. 3B). NoNR mutants were reported to exhibit chlorosis, which is the phenotype of bleached pigments on F2N plates containing nitrate as the sole nitrogen source8,9,12,17,21. Chlorosis is a typical phenotype of nitrate reductase mutants caused by depletion of the nitrogen source in N. oceanica. We used three plate types: F2N (normal F2N medium containing both nitrate and ammonium as the nitrogen source), F2N 50% seawater containing zeocin (F2N + Zeo), and F2N containing nitrate as the sole nitrogen source (F2N–NH4+). Because F2N medium has too high salt concentration for zeocin, an F2N 50% seawater medium containing zeocin plate was used as in previous reports22,23,24. The six tested strains with potential plasmid removal exhibited sensitivity to zeocin (F2N + Zeo) as observed for the wild-type, as well as chlorosis on the F2N–NH4+ plate, which is similar to the parental NoNR mutant strains harboring the all-in-one PtTALEN-ARS vectors. This result is consistent with our model (Fig. 1A), as the spot tests showed that the six tested strains exhibited loss of NoNR function, which is the TALEN target gene, and zeocin resistance gene, which is the antibiotic marker of the all-in-one PtTALEN-ARS vectors. Accordingly, we successfully obtained plasmid-removed NoNR mutant N. oceanica cells using all-in-one PtTALEN-ARS vectors.
Clearance of all-in-one PtTALEN-ARS plasmids from Nannochloropsis oceanica cells. (A) Workflow for the clearance of all-in-one PtTALEN-ARS plasmids from host cells. All-in-one PtTALEN-ARS plasmids were removed by using F2N without zeocin solid plate or liquid medium. AZ and BZ are collected single colonies that emerged in a selectable zeocin plate following the introduction of all-in-one NoNR PtTALEN-ARS vectors. (B) Verification of all-in-one PtTALEN-ARS plasmid clearance by PCR using primers for amplification of FokI nuclease of TALENs. All-in-one PtTALEN-ARS plasmids clearance of strains collected from each clearing process was analyzed by using FokI primers, the internal plasmid sequence. FokI: FokI PCR amplicons; TUB: tubulin-beta PCR amplicons; WT: wild-type genomic DNAs; AZ and BZ: total DNAs from a single colony of N. oceanica that emerged in a selectable plate by the introduction of all-in-one NoNR PtTALEN-ARS plasmids; AL and BL: total DNA from AZ or BZ treated for plasmid clearance as shown in this figure; A1-A12 and B1-B12: total DNAs from single colonies that emerged in a solid F2N plate by spreading the AL or BL culture solution; M: DNA ladder marker. (C) Verification of all-in-one PtTALEN-ARS vector clearance by PCR using four pairs of primers for the detection of the all-in-one PtTALEN-ARS vector. The plasmid clearance of strains that were not detected FokI PCR bands was verified again using the other 3 pairs of internal plasmid primers. PtTALEN-N: PtTALEN N terminal domain PCR amplicons; FokI: FokI PCR amplicons; KanR: kanamycin resistance gene PCR amplicons; ZeoR: zeocin resistance gene (ShBle) PCR amplicons; TUB: tubulin-beta PCR amplicons.
Verification of gene edited Nannochloropsis oceanica strains with vector removal. (A) NoNR target site sequences of strains with potential all-in-one NoNR PtTALEN-ARS plasmids removal. To rule out contamination with wild-type cells, the NoNR target site sequencing of potentially plasmid removed strains was performed. WT: Wild-type genomic DNAs; AZ and BZ: total DNAs from a single colony of N. oceanica that emerged in a selectable plate following introduction of all-in-one NoNR PtTALEN-ARS plasmids; A5, A7 and A11: total DNAs of clearance treated cells of AZ; B1, B2 and B3: total DNAs of clearance treated cells of BZ. Underlined sequences of WT strain indicate the TALEN binding sites. Dashes indicate deletions. (B) Verification of all-in-one PtTALEN-ARS vector clearance by phenotypic analysis using spot tests. Wild-type, TALEN strains, or potentially plasmid-removed strains were cultured with 2 mL F2N liquid medium, and 5 µL (3.6 × 104 cells µL−1) of resultant cultures were spotted onto solid plates. Because the spots of zeocin sensitive strains could not be seen on the F2N + Zeo plate in the picture with a white background, the F2N + Zeo plate pictures with a gray background are presented to show the spot marks. F2N: F2N plate; F2N + Zeo: F2N 50% seawater containing 2 µg L−1 zeocin plate; F2N–NH4+: F2N without ammonium plate.
The effects of all-in-one PtTALEN-ARS vectors on host cell growth and lipid accumulation (Supplementary Fig. S3) were also examined, because they are important for a molecular breeding tool. The NoNR frame shift mutants harboring all-in-one PtTALEN-ARS vectors (Strain ID: AZ and BZ) or the mutants removed the vectors (Strain ID: A5 and B1) were used. The lipid contents were assayed by TAG contents per culture and TAG contents per cell because both are important information for algal lipid production. No significant differences in growth among these strains were found at each time point. It was also found that the A5 strain accumulated significantly higher TAG contents than the parental strain, AZ. On the other hand, TAG contents were detected with no significant difference among WT, AZ, BZ, and B1 strains.
To establish the transgene-free genome editing system, electroporation using carrier-DNAs, which are transgenes, was inconvenient because these carrier-DNAs remain in the host cells even after plasmid removal. However, carrier-DNAs are necessary for measurable and efficient electroporation using the Gene Pulser II electroporator. Therefore, an electroporation system was established without carrier-DNAs using the highly efficient Elepo21 electroporator (Supplementary Table S3). We introduced the all-in-one TALEN-ARS vector into N. oceanica using the Elepo21. Under 1250–1750 V poring pulse voltage, approximately 1000 colony-forming units per µg DNA were obtained. In accordance with the vector-removable genome editing system and carrier DNA-free electroporation, we successfully established a transgene-free genome editing system.
Verification of removal efficiency for all-in-one PtTALEN-ARS plasmids
To efficiently remove the vectors, we verified the clearance method of all-in-one PtTALEN-ARS vectors. We postulated that the all-in-one PtTALEN-ARS vectors could be removed by a failure of inheritance at cell division or by endogenous degradation. First, we utilized liquid culture with large volumes (30 mL or 25 mL) of F2N medium, as shown in Supplementary Fig. S4A, expecting that the vectors would be eliminated by high cell division. The cells were collected from the large volume liquid F2N culture and confirmed plasmid clearance by PCR analysis (Supplementary Fig. S4B). However, in all tested cells, PCR bands were detected. Next, we attempted to cultivate strains harboring the all-in-one PtTALEN-ARS plasmids under phosphorus or nitrogen deficiency, as shown in Supplementary Fig. S5A, since these stress conditions may stimulate various endogenous degradation systems in Nannochloropsis species25. Total DNAs were extracted from cells and vector clearance was confirmed by PCR (Supplementary Fig. S5B). The results showed that PCR bands were also detected in all tested conditions. Finally, we attempted to isolate single cells using solid F2N plates, as shown in Fig. 4A, since it was judged that it would be difficult to completely eliminate cells harboring vectors from the cell population in which vectors were removed. Transformants were cultured in F2N liquid medium for 10 or 14 days and the resultant cultures were plated onto F2N solid plates. Single colonies were collected and total DNAs extracted from these colonies were used for PCR analysis (Fig. 4B). The results showed that PCR band intensities were substantially reduced in 8/12 (10 d) or 6/12 (14 d) of the tested colonies. We found that strain isolation using solid plates was essential for obtaining strains in which plasmids were removed.
Verification of the efficient clearance method for all-in-one PtTALEN-ARS plasmids from Nannochloropsis oceanica cells using solid F2N plates. (A) Workflow of the clearance of all-in-one PtTALEN-ARS plasmids from host cells using culture spreading onto solid F2N plates. All-in-one PtTALEN-ARS plasmids could be removed earlier than the procedure shown in the Fig. 2A workflow. (B) Verification of the clearance of all-in-one PtTALEN-ARS plasmids from host cells using culture spreading onto solid F2N plates by PCR. The 12 colonies that emerged in F2N without zeocin plates were collected, cultured with 2 mL F2N liquid medium, and used for the PCR analysis. WT: total DNAs extracted from wild-type cells; BZ: total DNAs extracted from individual colonies of all-in-one NoNR PtTALEN-ARS plasmids; 1–12: total DNAs extracted from strains collected from F2N solid plates; FokI: FokI PCR amplicons; TUB: tubulin-beta PCR amplicons; M: DNA ladder marker. 10 d and 14 d indicate the duration (10 or 14 days) of liquid culture.
