TALENs: Strategy and application in modern agriculture

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Indian Farmer
Volume 10, Issue 03, 2023, Pp. 125-129
Available online at: www.indianfarmer.net
ISSN: 2394-1227 (Online)
Original Article
TALENs: Strategy and application in modern agriculture
Poulomi Sen1, Umme Salma1, Avishek Chatterjee2, Suprabuddha Kundu2 and Jayita
Hore1
1Assistant Professor, School of Agricultural Sciences, Sister Nivedita University
2Assistant Professor, School of Agriculture, Swami Vivekananda University
Corresponding email Id: poulomi.s@snuniv.ac.in
Received: 21 March 2023 Published: 26 March 2023
Abstract
Genome editing has got a key breakthrough success in the era of molecular biology.
TALEN, one of the genome editing tools has played a major role in the advancements of
basic biology, reverse genetics, agriculture and clinical study. The first commercially
available edited crop variety was also developed through TALEN. The strategy relies on
specific gene knockouts by DNA strand break by restriction enzyme followed by DNA
repair system ultimately leading to predetermined site specific mutational changes.
Cytoplasmic gene editing, methyl sensitivity, less off targets and not requiring a known
Protospacer Adjacent Motif (PAM) has alleviated the drawbacks of another genome
editing tool, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9).
In different fields of agriculture especially in quality improvement of different crops
TALEN technique has got huge popularity.
Keywords: TALENs, DNA, Genome, mutation, DNA repair
Introduction
TALEN (Transcription Activator-like Effector nucleases) is a potential genome editing
technology that gives a diversified scope to target any DNA sequence in a wide range of
organisms. TALE (Transcription Activator-like Effector) protein was reported for the first
time in the plant pathogenic bacteria Xanthomonas spp. to alter the gene transcription of
the host plant to support virulence (Boch et al., 2009). For inoculation of TALE, a typical
type III secretion system is needed followed by colonization of bacteria in the host. TALE
can bind to any DNA sequence and when combined with nuclease it can cleave double
stranded DNA. This disruption is again followed by DNA repair through either non-
homologous end-joining (NHEJ) or homology directed repair (HDR) leading to induced
gene mutation by precisely altering the sequence. TALENs have been extensively used in
modifying plant genome for any kind of improvement. In this article we have tried to
briefly compile the general strategy and application of TALEN in modern agricultural
field.
1. TALEN construct
The commonly used TALEN system bears two TALEN units and a typical TALEN unit
comprises of DNA binding TALE repeat domains (arrays of highly conserved 30-33 amino
acid repeats) with flanking N and C terminal domains and one FokI nuclease catalytic
domain. Individual TALE repeat can bind to a single specific DNA sequence (A/G/T/C) but
the specificity is determined by two hyper variable amino acid residues present on
position 12 and 13 (Table 1; Streubel et al., 2012). These two variable positions are
known as Repeat Variable Diresidue (RVD). The conserved 30-33 amino acid repeat

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sequence is LTPDQVVAIAS**GGKQALETVQRLLPVLCQDHG where **denotes the RDV.
The length of the target site corresponds to the number of repeats of an array. A
thymine (T) is located at the 5' end precedes the initial base of TALE repeat.
Table 1: RDV of each TALE repeat domain with their DNA base specificity
NS
NN
NK
IG
NG
HD
NI
RDV
A/C/G/T
G/A
G
T
T
C
A
DNA base specificity
Each FokI nuclease catalytic domain attached to one TALE repeat domain finally
dimerizes to form the ultimate double stranded DNA breaks (DSBs) generating domain of
a final TALEN system (Fig. 1).
Fig 1: Schematic illustration of TALEN constructs and its function
2. Nuclease based DSBs and induced mutation
Each TALEN unit must bind specifically to the neighboring target DNA sequence with a
spacer region of 12-21 bases for proper nuclease induced double stranded DNA
cleavage. These DSBs are then followed by DNA repair system where DNA mutations like
insertion/deletion (INDEL) can be induced. In the presence of double stranded DNA
donor template, precise base substitution or insertion is commonly introduced. When two
nuclease based DSBs are placed on same chromosome, it leads to INDEL and if on
different chromosomes leads to chromosomal translocations conferring genome editing.
3. Benefits of TALENs
Base specificity of TALENs is highly accurate in the modern era of genome editing and
this helps the biologists to reduce the off target genome alterations. When it comes to
avoiding mutational changes to the genome, TALEN is considered as the most
dependable among the various genome editing techniques available because of its
degeneracy (certain RDVs can bind to more than one nucleotide).
TALENs give a powerful gateway towards epigenetic studies because of its methyl
sensitivity (Deng et al., 2012). Besides genomic DNA editing, TALENs have shown
perfect mitochondrial and plastid editing too in certain species (Piatek et al., 2018).
TALENs can efficiently regulate endogenous gene expression by developing artificial
transcription factors, repressors, activators. Targeted gene knock out in haploid cell by
TALEN is also possible followed by regenerating homozygous lines (Gurushidze et al.,
2014).
4. Delivery of TALENs in plant cells

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Depending upon the organisms, TALENs can be delivered into living cells by several
means like bacterial-based delivery (Agrobacterium-mediated transfer), physical
methods (Microinjection, particle bombardment, electroporation), viral based delivery,
chemical methods (PEG, liposomes etc.). However, for plant cell transformation in crops
like Rice, wheat, Arabidopsis, soybean, sugarcane, barley, tomato, potato, peanut,
Brassica sp. etc. Agrobacterium-mediated TALEN transfer and particle bombardment
have been extensively used.
5. Applications of TALENs in agricultural crops
TALENs have been significantly used for plant breeding for improving yield, biotic and
abiotic stress resistance, herbicide tolerance, nutritional quality improvement, better
shelf life. Few of the achievements are furnished below.
a. Biotic and abiotic stress resistance:
The earliest TALEN edited crop was Xanthomonas oryzae pv. oryzae (causing bacterial
leaf blight) resistant rice varieties. The gene OsSWEET14/Os11N3 was targeted for
TALEN based disruption (Li et al., 2012). Development of fungal disease powdery mildew
resistant barley and wheat by targeting the gene mlo1 was also possible by TALEN based
editing (Wang et al., 2014).
b. Quality improvement:
TALEN induced mutation in the gene producing the caffeic acid O-methyltransferase
(COMT) in sugarcane has improved its biofuel quality without compromising biomass and
stress resistance capacity (Jung et al., 2016). Two fatty acid desaturase genes were
mutated in soybean leading to reduced unhealthy trans-fat (linoleic acid) content and
increased oleic acid content and these were the first TALEN based genome edited
marketed varieties (Haun et al., 2014). Oleic content of peanut seeds was also improved
by TALEN induced mutation in the gene fatty acid desaturase 2(FAD2) (Wen et al.,
2018). Disruption of betaine aldehyde dehydrogenase 2 (BADH2) gene in rice through
TALEN tool has improved aroma of rice though enhancing the production of the major
fragrance compound 2-acetyl-1-pyrroline (Shan et al., 2015). Improvement of Nicotiana
benthamiana was done by developing plants which produce quality glycoproteins devoid
of plant-specific residues by knocking out two α-(1,3)-fucosyltransferase (FucT) and the
two β-(1,2)-xylosyltransferase (XylT) genes (Li et al., 2016). ZmMRP4 (Multidrug
resistance-associated protein 4), ZmIPK1A, ZmIPK, genes related to phytic acid (PA)
synthesis have been mutated by TALEN for improving nutritional quality of maize seeds
(Liang et al., 2014). Mutational change in ANT1 gene in tomato leads to a change in
anthocyanin accumulation in tomato tissue (Cermak et al., 2015). Reduction of
glycoalkaloid and cholesterol content of potato was made through TALEN induced
disruption in Sterol Side Chain Reductase 2 (StSSR2) gene (Zheng et al., 2021).
c. Phenotypic marker:
Knock out of the glossy2 (gl2) locus of maize conferred a glossy appearance (Si et al.,
2015). Gibberelic acid (GA) responsive tomato plants were produced by mutating the
DELLA protein producing gene Procera (pro) (Lor et al., 2014).
d. Yield:
Genes related to yield attributing parameters of rice like Dense and erect panicle 1
(OsDEP1), Cytokinin oxidase 2 (OsCKX2) were mutated by TALENs (Shan et al., 2013).
e. Herbicide resistance:
TALEN induced mutation in acetolactate synthase (ALS) genes led to resistance to
sulfonylurea and imidazolinone herbicide resistance in tobacco (Zhang et al., 2013).
Glyphosate sensitivity of rice has been changed by knocking out the gene 5-
Enolpyruvylshikimate-3-phosphate synthase (OsEPSPS) gene by this genome editing tool
(Wang et al., 2015).
f. Storage tolerance:
Mutation in vacuolar invertase gene (Vlnv) in potato has improved the chips colour by
reducing the acrylamide content and post-harvest shelf life (Clasen et al., 2015).
Storage tolerance of rice seeds can be improved through mutagenesis of lipoxygenase
(LOX3) gene using TALEN (Ma et al., 2015).
g. Mitochondrial genome editing:

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Cytoplasmic male sterility (CMS) was also overcome by TALENs induce mitochondrial
gene editing in rice and Brassica (Kazama et al., 2019).
h. Environment insensitivity:
Vernalization related gene FRIGIDA (FRI) in Brassica oleracea has been edited by
TALENs (Sun et al., 2013).
6. Conclusions
New Breeding Techniques (NBTs) like genome editing (cluster regularly interspaced short
palindromic repeats, CRISPR/Cas9, TALENs, zinc finger nucleases, ZNF), reverse
breeding, cisgenesis, site directed nucleases, homing endonucleases or meganucleases
etc. always offer additional benefits over the conventional procedures like selection or
selection accompanied with hybridization or mutational breeding or marker assisted
selection (MAS). Though CRISPR/Cas9 has tremendously helped breeders in improving
crops, TALEN has its own specific potentials that make it unique. It has high specificity,
low off target mutation, power to discriminate methylated and demethylated based and
even it can modify cytoplasmic genes too. The key disadvantage of TALEN is its tiring
construction and it can also be irrelevant if the objective can only be achieved by
TALENs. Optimizing the efficient TALEN delivery into the plant cell and use of fusion
proteins devoid of nuclease enzyme can be explored further.
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