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#ZFN

3 public questions tagged with this topic.

Transgenic Plant -2

Major advantage of CRISPR over ZFN/TALEN is:

Major advantage prompting global adoption of CRISPR over zinc finger nucleases and TALEN is unparalleled ease of design, cloning and deployment reducing cost and technical expertise barrier. ZFN construction involves library of zinc finger modules each specific for 5'GNN 3' triplet; assembly of three to six modules suffers from context dependence where finger-finger junctions alter specificity requiring selection via bacterial two-hybrid or OPEN platforms taking months and low success rates for AT-rich targets. TALEN improves modularity with simple RVD code but still demands assembly of 15-20

Ref: Hsu et al. Cell 2014 157:1262 CRISPR ease vs ZFN TALEN; Zhang Feng Nat Protocols ease design.

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Transgenic Plant -2

Like ZFN, TALENs use which nuclease domain?

Like zinc finger nucleases, transcription activator-like effector nucleases utilize catalytic domain of FokI endonuclease for cleavage because DNA binding domain lacks nuclease activity. TALE repeats provide programmable anchoring but cannot cut phosphodiester backbone; fusion via flexible linker to C-terminal 196 aa FokI cleavage domain confers scission capability. FokI domain requires dimerization, so two TALEN monomers engineered to bind opposite strands in tail-to-tail orientation with spacer 12 to 21 bp allow FokI domains to associate forming active nuclease complex generating double-stra

Ref: Christian et al. Genetics 2010 186:757 TALEN FokI; Joung Sander Nat Rev Mol Cell Biol 2013 FokI.

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Gene Therapy

CRISPR, ZFN and TALEN are used in gene therapy for:

CRISPR-Cas9, ZFN, TALEN constitute programmable site-specific endonucleases enabling precise genome manipulation. CRISPR uses single-guide RNA 20 nt spacer complementary to genomic target adjacent NGG PAM recognized Streptococcus pyogenes Cas9 possessing HNH domain cleaving complementary strand RuvC noncomplementary generating blunt double-strand break 3 bp upstream PAM. Zinc-finger arrays modules each recognizing 3 bp triplets linked FokI nuclease dimerize cutting within spacer, while TALEN repeats repeat-variable di-residues HD recognizing cytosine NI adenine NG thymine NN guanine provide si

Ref: NIH Genome Editing CRISPR ZFN TALEN Overview; Nature Biotech Comparison 2020; NCBI Gene Editing Therapy https://www.ncbi.nlm.nih.gov/books/NBK542207/.

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