
Transcription activator-like effector nucleases (TALENs) are fusion proteins, which combine a TAL effector DNA-binding domain and the DNA-cleavage domain of the FokI restriction enzyme. TALENs can introduce targeted DNA double-strand breaks (DSBs) in the plant genome (Christian
We not only face many technical challenges with genome-editing tools, but also public acceptance of the technology and government regulatory policies for its adoption. As of 2019, 24 gene-edited crops covering 16 crop species have been ruled outside the scope of traditional genetic-modification policies by the US Department of Agriculture. Cultivation of a high oleic acid soybean created by the Calyxt seed company using TALENs technology began in 2018, and its high oleic acid soybean oil was commercially marketed in the United States (reviewed by Park
Here, we report the development of
The TALEN-encoding binary vector pPZP-
A herbicide-resistant transgenic fixed rice line (Ba15) was used as the recipient variety for the TALEN vector transformation. The Ba15 rice line was developed by Dr. Myung-Ho Lim's group and confirmed to have a single copy of T-DNA harboring the CaMV 35S::
The sterilized mature rice seeds were sown on N6 medium containing 2,4-D herbicide in a dark room at 28℃. After four weeks, the compact embryogenic calli were transformed using a PDS1000/He particle bombardment system (Bio-Rad, Hercules, CA, USA) with a particle diameter of 0.6 mm and helium pressure of 1100 psi. The plasmid DNA of TALEN-encoding T-DNA binary vector (pPZP-
Genomic DNA was extracted from young leaves by the CTAB method. PCR strategies were designed for TALEN construct confirmation using two sets of PCR primers, namely a forward primer F1 (5′-CTACAGCGGTGGGTA CAATCT-3′) and reverse primer R1 (5′-CCCGATCTA GTAACATAGATGACAC-3′) for
The
For two transgenic
The platform-formatted microarray data processing was carried out on the CLC genomic workbench software, version 9.0 (www.qiagenbioinformatics.com/). Differentially expressed genes (DEGs) with an absolute value of Log2 fold-change > 2, and a false discovery rate (FDR)
The paired
To produce TALEN-based
To see whether the TALEN-mediated
In addition,
Table 1 . TALEN-mediated
Mutant lines | Generation | Mutation types | Mutation site (bp) | PAT expressionz) | Generation | Mutation sites (bp) | Amino acids | PAT expression | ||
---|---|---|---|---|---|---|---|---|---|---|
R6 | T0 | Start codon deletion/insertion | ‒1/‒3 | +/‒ | ‒/+ | T1 (R6,1-40) | +1/‒1/‒2/‒3 | Frame shift | +/‒ | ‒/+ |
SNP | A/G (4) | G/T (3), A/G (4), | Substitution | ‒ | ||||||
Stop codon deletion | ‒ | ‒2/‒3/‒4/‒5/‒8 | Deletion | + | ||||||
R9 | T0 | Start codon deletion/insertion | ‒1 | + | +/+ | T1 (R9, 1-31) | ‒1/‒3 | Frame shift | +/‒ | +, ‒/+ |
SNP | ‒ | G/T (3), G/A (22), | Substitution | ‒ | ||||||
Stop codon deletion | ‒1 | ‒1/‒4/‒5 | Deletion | + |
z)Active (+) and inactive (−) PAT protein expression.
y)Presence (+) and absence (−) of
x)Underlined characters indicate mutations in the TALE:FokI cleavage site.
We selected three T1 plants of R6-2, R9-15, and R9-20 with complete disruption of PAT protein expression and high seed-setting rates for T2 plant productions. Twenty-seven of T2 seeds derived from R6-2, 23 seeds from R9-15, and four seeds from R9-20 were sown in the greenhouse. We analyzed PAT protein expressions by immuno-strip and results indicated that these 54 of the T2 plants had successfully inactivated
To obtain rice lines harboring the
Using microarray analysis, transcriptome profiling was performed on three transgenic rice lines (R6-2-4, R9-15-4, and R9-20-2) and the recipient variety, the herbicide-resistant transgenic rice Ba15. The three lines were confirmed to be TALEN-DNA-free and to have inactive
Gene set enrichment analysis revealed 72 enriched DEGs that were classified into the two GO terms “cellular component” and “molecular function.” In the cellular component category, the GO term “plasma membrane” (GO: 0005886) involved 20 DEGs, including 10 upregulated and 10 downregulated DEGs with the highest enrichment scores. Sixteen DEGs (10 upregulated and 6 downregulated DEGs) that belonged to the GO term “extracellular region” (GO: 0005576) showed significant enrichment scores. In the molecular function category, the most significantly enriched GO term was “response to polysaccharide binding” (GO: 0030247) involving five DEGs comprised of four upregulated and one downregulated DEG (Table 2 and Table 3). Furthermore, DEG analysis revealed significantly downregulated
Table 2 . Gene Ontology (GO) annotation and GO enrichment analysis at
Gene Ontology | Terms | Cluster frequencyz) | |
---|---|---|---|
Cellular Component (CC) | Plasma membrane (GO:0005886) | 27.78% | 0.003531717 |
Extracellular region (GO:0005576) | 22.22% | 0.004769093 | |
Molecular Function (MF) | Polysaccharide binding (GO:0030247) | 6.94% | 0.00029600 |
z)Cluster frequency was calculated as ratio of enriched gene numbers of each term to a total of 72 DEGs that were enriched for GO terms.
Table 3 . Enriched DEGs detected from
SEQ_ID | Log2 fold changes | Descriptions | GO termsz) | TAIRy) | ||
---|---|---|---|---|---|---|
R9-15-4 | R9-20-2 | R6-2-4 | ||||
−12.263 | −12.419 | −13.009 | - | TALEN-target | ||
Os03t0392600-01 | −9.128 | −8.358 | −8.999 | Peptidase S10, serine carboxypeptidase family protein | CC | AT2G27920 |
Os09t0358000-00 | −8.633 | −6.202 | −8.516 | Similar to OsD305 | CC | AT1G51890 |
Os11t0695000-01 | −7.744 | −7.639 | −6.600 | Similar to Leucine Rich Repeat family protein | CC | AT3G47570 |
Os09t0356800-01 | −7.271 | −3.446 | −7.168 | Protein kinase, core domain containing protein | CC | AT1G51850 |
Os11t0514500-01 | −6.766 | −8.222 | −6.396 | Sorghum bicolor leucine-rich repeat-containing extracellular glycoprotein precursor | CC | AT5G21090 |
Os02t0483000-00 | −6.648 | −5.603 | −5.616 | Similar to fasciclin-like arabinogalactan protein 8 | CC | AT3G12660 |
Os11t0641500-01 | −4.364 | −4.436 | −5.929 | Cupredoxin domain containing protein | CC | AT3G09220 |
Os03t0184550-01 | −3.596 | −2.900 | −2.921 | Similar to Dihydroflavonol-4-reductase | CC | AT4G33360 |
Os10t0343400-01 | −3.207 | −3.590 | −2.886 | Cellulose synthase family protein | CC | AT3G03050 |
Os10t0142600-00 | −2.762 | −3.686 | −4.315 | Protein kinase, catalytic domain domain containing protein | CC, MF | AT1G21270 |
Os02t0740700-01 | −2.512 | −3.015 | −2.516 | Peptidase M10A and M12B, matrixin and adamalysin family protein | CC | AT1G24140 |
Os11t0115350-01 | −2.227 | −3.853 | −3.681 | Similar to Non-specific lipid-transfer protein 2 | CC | AT2G38540 |
Os12t0228700-00 | −2.156 | −3.158 | −3.029 | Similar to 32 kDa protein | CC | AT1G73040 |
Os04t0175600-01 | 2.005 | 2.702 | 2.029 | Similar to o-methyltransferase (EC 2.1.1.6) (Fragment) | CC | AT5G54160 |
Os12t0583300-01 | 2.390 | 2.923 | 2.099 | Peptidase aspartic, catalytic domain containing protein | CC | AT2G03200 |
Os05t0318700-01 | 2.527 | 3.501 | 4.265 | Similar to Resistance protein candidate (Fragment) | CC | AT3G51550 |
Os09t0339000-01 | 2.718 | 4.084 | 2.400 | Protein kinase, core domain containing protein | CC | AT5G10530 |
Os11t0605100-01 | 3.197 | 4.683 | 5.049 | NB-ARC domain containing protein | CC | AT3G14470 |
Os02t0111600-01 | 3.245 | 3.698 | 2.199 | Serine/threonine protein kinase-related domain containing protein | CC, MF | AT1G21230 |
Os12t0431100-01 | 3.366 | 2.913 | 2.211 | ATPase, AAA-type, core domain containing protein | CC | AT5G40010 |
Os07t0539900-01 | 3.497 | 3.157 | 2.956 | Similar to Beta-1,3-glucanase-like protein | CC, MF | AT4G26830 |
Os01t0944500-00 | 3.682 | 3.588 | 3.094 | Glycoside hydrolase, family 17 domain containing protein | CC, MF | AT3G57240 |
Os01t0660200-01 | 3.759 | 4.487 | 2.663 | Acidic class III chitinase OsChib3a precursor (Chitinase) (EC 3.2.1.14) | CC | AT5G24090 |
Os06t0566300-00 | 3.867 | 2.056 | 2.389 | Similar to zinc transporter 4 | CC | AT1G10970 |
Os01t0713200-01 | 4.312 | 3.960 | 2.744 | Similar to Beta-glucanase | MF | AT3G57260 |
Os06t0143950-00 | 4.396 | 5.334 | 6.007 | Non-protein coding transcript | CC | AT1G79990 |
Os02t0550800-01 | 4.446 | 4.496 | 3.998 | Ammonium transporter family protein | CC | AT2G38290 |
Os12t0628600-01 | 4.760 | 3.073 | 2.335 | Similar to Thaumatin-like pathogenesis-related protein 3 precursor | CC | AT4G11650 |
Os07t0131375-00 | 6.228 | 5.106 | 5.720 | Protein kinase, catalytic domain domain containing protein | CC | AT2G37710 |
Os11t0214700-00 | 7.629 | 3.431 | 3.181 | Plant disease resistance response protein domain containing protein | CC | AT5G42510 |
Os01t0382000-01 | 8.453 | 6.368 | 6.203 | Similar to Pathogenesis-related protein PRB1-2 precursor | CC | AT4G33720 |
z)CC, cellular component; MF, molecular function.
y)Means The Arabidopsis Information Resource.
In 2018, the first TALEN-edited high oleic acid soybean with approximately 80% oleic acid, which had been developed by Calyxt in the USA, was cultivated, and high oleic soybean oil was successful commercialized and released on the US market (Reviewed in Park
In this report, we identified some challenging problems. First, we generated 41 T0 plants comprised of 14 lines with
Second, most mutations in the T0 and T1 plants occurred in non-target sites of the
Finally, many
Many studies have focused on the possible occurrence of off-target edits in mutant plants edited by TALENs or the CRISPR/Cas9 system, which have been reported to occur at a low frequency in plants (Zhang
This work was supported by a grant (PJ01432206 and PJ01194401) from the National Institute of Agricultural Sciences (Rural Development Administration, Republic of Korea).
The authors declare that there is no conflict of interest.
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