Zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALEs) are genome editing tools that use engineered DNA-binding domains to target specific locations in the genome. ZFNs use zinc finger proteins fused to the FokI nuclease, while TALEs contain repetitive sequences that determine nucleotide specificity. Both tools can be used to create targeted double-strand breaks, enabling precise genome editing through non-homologous end joining or homology-directed repair. The document further describes various applications of ZFNs and TALEs in biomedical research such as nucleases, recombinases, transposases, and artificial transcription factors.

1. GENOME EDITING TOOLS
ZFNs and TALEs
MANITA PANERI
PhD Scholar
Medical Microbiology

2. How Does Genome Editing Work?
Genome editing is a process where an organism’s genetic code is changed. Scientists use
enzymes to ‘cut’ DNA creating a double-strand break (DSB). DSB repair occurs by non-
homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ produces
random mutations (gene knockout), while HDR uses additional DNA to create a desired
sequence within the genome (gene knock-in).

3. ZINC FINGER PROTEINS
• A zinc finger is a small protein structural motif that is
characterized by the coordination of one or more zinc ions
in order to stabilize the fold.
• Xenopus laevis TFIIIA was originally demonstrated to
contain zinc and require the metal for function in 1983, the
first such reported zinc requirement for a gene regulatory
protein.

4. Classification:
ZINC FINGER
PROTEIN
Cys₂His₂ Gag knuckle Treble clef Zinc ribbon Zn₂/Cys₆

6. Cys₂His₂:
• The Cys₂His₂- like fold group is the best characterized
class of zinc fingers, and is common in mammalian
transcription factors.
• This class of zinc fingers can have a variety of functions
such as binding RNA and mediating protein-protein
interactions but it is best known for its role in sequence-
specific DNA-binding proteins such as Zif268.
• The alpha helix of each domain can make sequence-
specific contacts to DNA bases.

7. Gag knuckle:
• This fold group is defined by two short beta strands
connected by a turn(zinc knuckle) followed by a short
helix or loop and resembles the classical Cys₂His₂ motif
with a large portion of the helix and beta hairpin
truncated.
• The retroviral nucleocapsid (NC) protein from HIV and
other related retroviruses are examples of protein
possessing these motifs.

8. Treble-clef:
The treble-clef motif consists of a beta hairpin at N-terminus and an
alpha-helix at the C-terminus that each contribute two ligands for
zinc binding, although a loop and a second beta hairpin of varying
length and conformation can be present between the N-terminal
beta hairpin and the C-terminal alpha helix.
These fingers are present in a diverse group of proteins that
frequently do not share sequence or functional similarity with each
other.
The best characterized proteins containing treble-clef fingers are
the nuclear hormone receptors.

9. Zinc ribbon:
The zinc ribbon fold is characterised by two beta hairpin
forming two structurally similar zinc-binding sub-sites.

10. Zn₂/Cys₆
• This class contain a binuclear zinc cluster in which
two zinc ions are bound by six cysteine residues.
• These zinc fingers can be found in several
transcription factors including the yeast Gal4
protein.

11. Application of zinc finger proteins in biomedical research
• Zinc finger nucleases
• Zinc finger recombinases
• Zinc finger transposases
• Zinc finger artificial transcription factor
• Protein delivery
• DNA diagnostics

12. ZFNs ( ZINC FINGER NUCLEASES)
• Zinc finger nucleases (ZFNs) are a class of
engineered DNA-binding proteins that
facilitate targeted editing of the genome by
creating double-strand breaks in DNA at user-
specified locations. These were first
endonucleases that are based on zinc finger
proteins, a family of naturally occurring
transcription factors, fused on an
endonuclease FokI.

13. Zinc finger recombinases (ZFRs)
Like ZFNs, they consist of two inverted ZFs on double-stranded DNA with the recombinase domain exercising its catalytic activity in the
20 bp central flanking region. Successful re-engineering of serine recombinases was studied to explore the specificity and effectiveness
of ZFRs. Using this approach, Gosh. et al generated enhanced hybrid recombinases based on activated catalytic domains derived from
the resolvase/invertase family of serine recombinases .
The re-engineered hybrid recombinases showed higher specificity with low toxicity, indicating the potential of these enzymes in a wide
range of applications for genome engineering and gene therapy.
Zinc finger transposases
The Sleeping Beauty (SB) transposon is an integrating vector system capable of inserting expression cassettes with high stability. Fusion
of a ZF with SB transposase resulted in a fourfold enrichment of the transposon insertion as compared to native SB transposase.
Zinc finger-artificial transcription factor (ATF)
DNA-binding domains including ZFs can be engineered to regulate expression of specific genes by fusing them to transcriptional or
epigenetic effector domains, thus generating artificial transcription factors (ATFs). In principle, ATFs are comprised of a DNA-binding
domain, a transcriptional activator (VP16 and p65 domains) or repressor (KRAB domain), and a nuclear localization signal (NLS) to
ensure the efficient transport of ATFs into the nucleus. Engineered ZFPs were fused to VP64 and KRAB domains to create synthetic
activators and repressors, respectively.

14. Protein delivery
ZFNs are intrinsically cell permeable which is attributed to the net positive charge of ZF domains. Cell penetrating
ZF domains were successfully proven to be good protein transduction reagents. Gaj et al. demonstrated that when
the N-terminus of firefly luciferase was genetically fused to two or three fingers, it resulted in cell penetrating
properties as effective as Lipofectamine-mediated plasmid transfection. These protein-fused ZFs are capable of
delivering functional proteins into primary and transformed mammalian cells.
DNA diagnostics
A system called Sequence Enabled Reassembly of β-lactamase (SEER-LAC) consists of two split enzymatic domains
of β-lactamase that would reassemble into a full-length enzyme upon ZFPs binding to their target DNA. Kim et al.
(2011) developed a ZFP array combined with the SEER-LAC system for DNA diagnostic applications. The ZFP array
with the SEER-LAC system generated DNA-dose dependent signals with a visual readout and allowed for a
quantitative assay. Their result suggested the potential use of this system to develop a point-of-care (POC)
diagnostic for pathogen detection.

15. TALEs Protein

16. • Transcription activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria to aid the
infection of plant species. TALEs assist infections by binding to specific DNA sequences and activating the
expression of host genes.
• They recognize plant DNA sequences through a central repeat domain consisting of a variable number of 34
amino acid repeats.
• These proteins are interesting to researchers both for their role in disease of important crop species and the
relative ease of retargeting them to bind new DNA sequences.
• The crystal structure of a TALE bound to DNA indicates that each repeat comprises 2 alpha helices and a short
RVD-containing loop where the second residue of the RVD makes sequence specific DNA contacts while the
first residue of the RVD stabilizes the RVD-containing loop.
• The target sequence of all naturally occurring TALEs begins with a thymine (T) nucleotide at 5’end, which is
important for the functionality of TALE’s activity.

17. TALEs have a modular DNA-binding domain (DBD) consisting of repetitive sequences of residues; each repeat
region consists of 34 amino acids.
A pair of residues at the 12th and 13th position of each repeat region determines the nucleotide specificity and
are referred to as the repeat variable di-residue (RVD). The last repeat region, termed the half-repeat, is typically
truncated to 20 amino acids. Combining these repeat regions allows synthesizing sequence-specific synthetic
TALEs.
The C-terminus typically contains a nuclear localization signal (NLS), which directs a TALE to the nucleus, as well
as a functional domain that modulates transcription, such as an acidic activation domain (AD).

18. Application of TALEs in Biomedical Research
1. TALE nucleases: TALENs are designed in pairs to make contact with the two opposing strands of the target DNA, separated
by a spacer to provide the FokI nuclease domains with enough space to dimerize and create a DNA DSB.
2. TALE recombinases : The first attempt to generate a chimeric TALER was carried out by Barbas’s group. They created a
library of truncated TALE variants to identify optimized TALER fusions with a catalytic domain from the DNA invertase from
Gin. Their study showed that TALERs can be used to recombine any DNA sequence in bacteria and mammalian cells, which
may overcome the limitation of the modular targeting capacity of ZFRs. They also demonstrated the reprogram ability of
the recombinase’s catalytic specificity.
3. TALE transposases: The non-viral PB transposable element fused with the Gal4 DBD has been studied to address the
problem of integrating viral vectors associated with insertions at unwanted sites. Owens et al., generated hyperactive PB
transposases fused with custom-designed TALEs to target the first intron of the human CCR5 gene. They have
demonstrated targeted transposition to the CCR5 genomic safe harbor, which allows for stable expression of a transgene
across multiple cell types.
4. TALE-artificial transcription factors: Engineered TALEs can be fused to transcriptional activator and repressor domains to
construct artificial transcription factors (ATFs). TALE-ATFs have been successfully used as gene-specific activators and
repressors. Engineered TALEs fused with a VP64 domain were shown to target a wide spectrum of DNA sequences at a
similar or greater level compared to ZF-ATF bearing a VP64 domain.