The document discusses TALENs (Transcription Activator-Like Effector Nucleases) as a modern tool for genome editing in plants, emphasizing their development and application in genetic engineering. It outlines the mechanisms of genetic recombination, DNA restriction and repair, as well as the engineering of TALENs to target specific DNA sequences for gene modification. The document serves as an educational resource, detailing the process of constructing and utilizing TALENs for gene deletions and insertions within plant genomes.

1. TALENS
GENOME EDITING IN PLANTS WITH TALENS

2. Introduction
Genome editing is one of the most important tools which
supports genetic engineering. It is based on the naturally
occurring mechanism of DNA recombination which
involves the initiation of breaks with the double stranded
DNA followed by repair by the endogenous DNA
polymerases.
Conventional techniques such as gene knockouts using P-
elements and transposable genetic elements have been
superseded by more accurate genome editing methods
such as TALENs and CRISPR/Cas.

3. Objectives
This module has been designed to:
1. Introduce the participants to the concept of
genetic recombination and NHEJ repair.
2. Describe the development and application of
TALENs.

4. Learning Outcomes
Upon completion of this module, you should be
able to:
1. Describe the mechanism of genome editing.
2. Describe the principle and application of
TALENs to plant genetic engineering.

5. How do genetic engineers utilize
naturally occurring processes of DNA
Replication and Repair to edit the
genome?
THE PRINCIPLE

6. Genome Editing: Three steps
 Target the locus.
 Delete or insert a gene.
 Repair the DNA strand.

7. What is Genome Editing based on?
 The genetic material in every living cell is being
constantly subjected to forces which drive genetic
recombination and repair. Homologous
recombination is driven by DNA modifying
enzymes which include Restriction
endonucleases, Recombinases, Polymerases and
Ligases.
 Genome editing harnesses these natural process
to achieve the objective of modifying the genome
in a precise and reproducible manner.

8. Process 1: Recombination
 Genetic recombination occurs during meiosis when
genetic material is exchanged during the process of
homologous recombination.
 The enzyme DNA recombinase initiates this process by
catalyzing directionally sensitive DNA exchange
reactions between short (30–40 nucleotides) target site
sequences that are specific to each recombinase.

9. Process 1: Recombination
RecA (bacterial)/ Rad51(H. sapiens) are
involved in homologous repair and
recombination in double stranded DNA break
repair. Rad51 protein, performs the tricky task
of pairing up a broken strand with its backup
copy. The broken strand is first processed by
other proteins to expose a single strand. Rad51
surrounds this single strand, and then captures
the backup DNA copy, matching the sequence
of the broken strand with a homologous
sequence in the intact DNA double helix. Inside
the Rad51 complex, the single strand is then
exchanged for one of the strands in the duplex
DNA, powered by ATP. Finally, a host of other
proteins fill in all the missing sections of DNA,
ultimately restoring two matching copies.

10. Process 2: DNA Restriction
Restriction endonucleases ‘cut’
or ‘restrict’ the genome by
identifying a specific set of
sequences within the genome.
In living bacteria these
enzymes serve as a means to
defend the cell against
invading bacteriophages. The
restriction sites within the host
genome are heavily
methylated, this protects the
host genome from digestion. A
bacterium is immune to its own
restriction enzymes.

11. Process 2: DNA Restriction
 How can restriction enzymes be applied to
genome editing in vivo?
The fragment of the restriction enzyme that is involved in
the cleavage of the DNA molecule can be fused with
Transcription Activator (TAL) proteins in order to develop
TALENs.

12. Process 3: DNA Repair
 DNA polymerases play a
critical role in repair of the
genome after the
insertion or deletion of a
gene irrespective of the
genome editing strategy
which is employed.
 DNA Ligases are involved
in non-homologous
recombination which
occurs when genes are
deleted.

13. Engineering of DNA binding proteins.
Genome Editing Tools: TALENs

14. The Discovery of TAL
 Transcription Activator Like Effectors (TAL).
 Discovered in Xanthomonas.
 Injected into plant cells via the Type III
secretion systems.
 Bind to promoter sequences of genes which
facilitate bacterial infection.
 Specific binding to the DNA is based on a 34
amino acid repeat motif.

15. How do TALs function?
TA
L
Proteins which facilitate
infection

16. Typical Structure of TALs
 Central repeat domain.
 Number of repeats 1.5 to 33.5
 Each repeat consists of 34 residues.
 Typical repeat sequence:
LTPEQVVAIASNGGGKQALETVQRLLPVLCQAH
G
 Short repeat at the Carboxyl terminal end.
 Hypervariable residues: position 12 and 13.
(Repeat Variable Domain).
 Structure: two alpha helices and a short RVD.

17. Typical Structure of TALs
LTPEQVVAIASNGGGKQALETVQRLLPVLC
QAHG
This thirty four amino acid domain is repeated (1.5) to
(33.5) times within a typical TAL.

18. Typical Structure of a TAL
Amino Acid Symbol
Asparagine N
Histidine H
Glycine G
Isoleucine I
Aspartic acid D

19. How do we engineer TALs?
 Combinations of amino acids bind to specific
nucleotides.
 This code has been deciphered.
 It is now possible to engineer TALs to target
specific DNA sequences in vivo and in vitro.
 TALs can be fused to functional domains and
applied to edit the genome.

20. TAL: Amino Acid – Nucleotide
5’ – T G C A – 3’
NN NI

21. TALENs
Deciphering the TAL binding sequences led to the first patent on TALEs filed
by the University of Harvard.

22. Constructing a TALEN
 TALENs (Transcription Activator Like Effector
Nucleases)
 TAL = DNA Binding Domain.
 EN = Endonuclease of other DNA modifying
domain.
 The endonuclease domain from the enzyme
FokI derived from the bacterium
Flavobacterium okeanokoites.

23. Structure of a TALEN
Engineered DNA binding Motifs
FokI
nuclease
The DNA binding motifs are engineered based on the coding amino acids in
the TAL motif. The DNA sequence of the restriction enzyme FokI is fused to
the DNA sequence for the TAL repeats resulting in the creation of a chimeric
enzyme which can target specific regions of the DNA.

24. Engineering TALENs
Identify the specific region of the DNA which you intend to edit.
Design the sequence of the TAL domain based on the TAL code.
Inset the target gene into a TAL plasmid
Transform the TAL plasmid into the host and assay for specificity of
deletion /insertion using OCR or phenotypic screening.

25. How do TALENs work?
A pair of TALENs, one specific to each region flanking the targeted locus
(black) are transfected into the cell and expressed in vivo. Upon expression
the FokI domain restricts the DNA on either side of the locus resulting in a
double stranded break.

26. How do TALENs work: Deletions
The DNA repair mechanism attempts to repair the gap by a process known
as gap filling. It recruits DNA polymerases and DNA repair enzymes.
However in the absence of either of the template strands, the gap is
repaired without the gene. This results in a gene deletion.

27. How do TALENs work: Insertions
If we introduce a DNA fragment with a homologous recombination site that
matches the regions flanking the deletion, the DNA fragment integrates into
the double stranded break. This results in a gene insertion.

28. Experimental Design
1. Construct a TALEN
2. Transform into the host cell
3. Induce the expression of the TALEN
4. Verify the gene knockout by PCR or
phenotypic screening.

29. Experimental Design
Apply what you have just learnt in this
module to resolve a specific problem.

30. Applying what you have learnt..
How will you apply what you have learnt to
delete the following gene using a TALEN
mediated approach?
Reference Sequence: EF139435