#ZFNs

Transgenic Plant -2

Genome editing method most precise at predetermined site is:

Precision of genome editing methods varies drastically correlating with targeting mechanism. Random mutagenesis approaches such as TILLING based on EMS alkylation of guanine causing G to A transitions genome-wide at ~1 per 150 kb requires screening massive populations via CEL1 nuclease assay and mapping; transposon mutagenesis with Ac/Ds or Mutator inserts at TA dinucleotides semi-randomly within genes, excision leaves footprint and instability; T-DNA insertion via Agrobacterium integrates preferentially into euchromatic gene rich regions at 5'-UTRs causing large deletions and chromosomal rearrangements. All three generate unpredictable background mutations complicating breeding. CRISPR/Cas9 provides defined precision at predetermined site because single guide RNA 20 nt dictates cleavage 3 bp upstream of PAM NGG via Watson-Crick base pairing, enabling base editors fusing deactivated Cas9 to deaminases for A to G or C to T changes without DSB, prime editors for insertions. Multiplexing with several guides edits multiple loci simultaneously without crossing. Whole genome sequencing confirms minimal off-targets when high-fidelity Cas9 variants like SpCas9-HF1 employed. This site-specific precision revolutionized crop trait stacking and therapeutic editing for sickle cell disease correcting point mutation.

Ref: Doudna Charpentier Science 2014 CRISPR precision; Voytas Plant Cell 2013 Editing comparison.

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Transgenic Plant -2

Most commonly used RVDs include NI, HD, NG and NN which recognize:

Four repeat variable diresidues commonly used in TALEN assembly define simple cipher: NI comprising asparagine isoleucine preferentially binds adenine forming hydrogen bond between Asn and N7, HD histidine aspartate specifies cytosine via Asp carboxyl hydrogen bond to cytosine exocyclic amine and salt bridge, NG asparagine glycine recognizes thymine via van der Waals contacts between glycine carbonyl and methyl group at 5 position, NN asparagine asparagine tolerates guanine and adenine due to bulky purine accommodation but later improved by NK asparagine lysine or NH asparagine histidine more specific for guanine. Thus to target sequence A C T G one would arrange NI HD NG NN respectively. Target design typically requires preceding T at position 0 due to N-terminal cryptic repeat. Golden Gate assembly uses RVD modules as building blocks for custom TALE genes 18 repeats long yielding specificity approaching single locus in genome. Variants include N* recognizing 5-methylcytosine enabling methylation-sensitive editing, and combination of RVDs allow recognition of virtually any DNA sequence except problematic homopolymeric runs that reduce affinity. This RVD-nucleotide dictionary transformed programmable DNA binding beyond zinc fingers.

Ref: Cermak et al. Nucleic Acids Res 2011 39:e82 RVD NI HD NG NN; Bogdanove Voytas Science 2011.

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Transgenic Plant -2

ZFNs induce genome modification by creating:

Zinc finger nucleases induce targeted genome modification by introducing double-strand breaks which are highly recombinogenic lesions activating cellular DNA damage response. After FokI dimer cleavage within spacer producing 5' overhangs, MRN complex Mre11-Rad50-Nbs1 senses ends, recruits ATM kinase phosphorylating H2AX, mediators 53BP1, initiating cell-cycle checkpoint. Breaks must be repaired to avoid apoptosis. In absence of donor, classical NHEJ mediated by Ku70/Ku80 heterodimer binding ends, DNA-PKcs recruitment, Artemis processing and Lig4-XRCC4 ligation rejoins ends frequently introducing small insertions or deletions due to processing, causing frameshift-mediated knockout useful for disrupting negative regulators like MLO mildew susceptibility in barley. If homologous donor plasmid with homology arms flanking break provided in excess during S/G2 phase, Rad51 mediated homologous recombination uses donor as template copying desired edits resulting precise gene replacement or insertion. DSB stimulates HR frequency up to 1000-fold over spontaneous. Detection of editing uses Surveyor assay, T7E1 cleavage, deep amplicon sequencing showing indel signatures distinct for each repair outcome.

Ref: Jasin Rothstein CSH Perspect 2013 DSB repair; Carroll Genetics 2011 ZFN breaks DSB.

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Transgenic Plant -2

Zinc finger nucleases (ZFNs) consist of:

Zinc finger nucleases represent first generation programmable nucleases fusing custom DNA recognition to cleavage effector. Each ZFN monomer architecture includes tandem array of Cys2-His2 zinc finger motifs originally derived from transcription factor Zif268 and Sp1, each finger ~30 amino acids folded into beta-beta-alpha structure where Zn2+ tetrahedrally coordinated by two cysteines in beta hairpin and two histidines in alpha helix stabilizing domain. Recognition helix positions -1 to 6 contact major groove bases via hydrogen bonds and van der Waals contacts. Array of three to six fingers recognizes 9 to 18 base pairs contiguously. C-terminal FokI endonuclease domain from Flavobacterium okeanokoites comprises nonspecific cleavage domain 196 amino acids requiring dimerization for double strand break formation leaving 4 bp 5' overhangs. Flexible linker GSGGS between finger array and FokI permits optimal spacing. Pair of ZFNs binding opposite strands tail-to-tail spaced 5-7 bp allows FokI dimer formation cutting intervening sequence, creating DSB stimulating homologous recombination or mutagenic NHEJ for targeted knockout or knockin applications in crops and human therapeutics for CCR5 editing and hemophilia correction.

Ref: Kim et al. PNAS 1996 ZFN; Carroll Annu Rev Biochem 2014 ZFN; Chandrasegaran 2016.

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Gene Tar getting

Which feature is common between TALENs and ZFNs?

Zinc finger nucleases and transcription activator-like effector nucleases are chimeric nucleases sharing cleavage module. Both fuse sequence specific DNA binding domain to non-specific FokI endonuclease catalytic domain from Flavobacterium okeanokoites. FokI must dimerize to cut DNA, so two ZFN or TALEN monomers bind opposite strands with spacer. DNA binding domains differ: zinc fingers versus TALE repeats, but nuclease domain identical. CRISPR instead uses Cas9 nuclease. Conservation of FokI domain explains similar requirements for FokI dimerization and spacing rules in both platforms.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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