Transgenic Plant -2

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 repeat plasmids via Golden Gate requiring multiple ligations, sequence verification, 5-7 days per TALEN, limited by repeat instability in E. coli due to homologous recombination. CRISPR eliminates protein engineering entirely: chemically synthesized pair of oligonucleotides encoding 20 nt spacer annealed and ligated into guide RNA expression vector or synthesized as single guide RNA ribonucleoprotein complex ready for transfection electroporation within one day. No protein evolution. Commercial kits provide optimized SpCas9 protein, high-fidelity variants, base editors. Consequently labs worldwide adopted CRISPR for gene knockout, knockin, activation, leading to over 10000 publications yearly, crop edits and two Nobel prize.

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

CRISPR/Cas9 specificity is provided by:

Specificity of CRISPR Cas9 system is dictated predominantly by guide RNA rather than protein itself, contrasting with zinc finger and TALEN protein-guided recognition. Cas9 from Streptococcus pyogenes contains recognition lobe and nuclease lobe with HNH and RuvC domains, plus PAM interacting domain at C-terminus that scans DNA for 5'-NGG-3' PAM via major groove interactions, required for binding. Spontaneous DNA unwinding initiates RNA-DNA heteroduplex formation with 20 nt spacer of crRNA embedded in single guide RNA fusion with tracrRNA scaffold. Complementarity drives conformational activation: full pairing especially in seed region nucleotides 1-8 proximal to PAM triggers HNH domain rotation cutting target strand complementary, RuvC cutting noncomplementary strand. Mismatches in PAM distal region tolerated leading to off-targets predictable by computational tools. Therefore reprogramming requires only swapping 20 nt spacer chemically synthesized within hours, whereas Cas9 protein unchanged. This RNA-guided programmable nature underlies ease and multiplex ability simply expressing multiple guides, enabling library screening, lineage tracing and epigenome editing fusing dCas9 to effectors where guide directs localization.

Ref: Jinek et al. Science 2012 337:816 Cas9 guide RNA; Nishimasu Cell 2014 156:935 specificity.

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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

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-strand break with variable overhang within spacer. Truncation analysis identified optimal N-terminal 152 aa upstream of repeats and C-terminal 63 aa linker downstream essential for activity, influencing binding affinity and steric positioning of FokI. Introduction of heterodimeric FokI mutations ELD KKR improves specificity reducing homodimer off-target cutting. Use of shared FokI domain enables transfer of knowledge about dimerization requirement, spacer length optimization, and fidelity enhancements from ZFN to TALEN, streamlining both platforms and establishing architecture reused in dimeric CRISPR-FokI dCas9-FokI fusions for improved specificity via dual guide requirement.

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

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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

TALE repeat variable diresidues (RVDs) recognize:

TALE repeat specificity is encoded by repeat variable diresidues RVDs located at positions 12 and 13 within each 33-35 aa repeat. Repeats fold as two helix bundle forming superhelical structure wrapping major groove around B-form DNA in contiguous tracking left-handed. Crystal structure solved 2012 shows each repeat contacts single base pair phosphate backbone via conserved residues while RVD at tip reads base via side chain interactions: hydrogen bonding and steric complementarity. Unlike zinc fingers recognizing triplets with neighbor influence, one repeat one base makes code highly modular and predictable enabling assembly of arrays recognizing 15-20 bp with straightforward cloning. N-terminal noncanonical repeats 0 and -1 contact 5' T required for optimal binding due to cryptic repeat. Some RVDs tolerate degeneracy broadening possibilities but overall specificity high. This modularity allowed Golden Gate assembly kits where RVD modules multimerized into full TALE in single reaction within days facilitating high-throughput genome editing in zebrafish, human pluripotent stem cells and tomato, overcoming context dependence bottleneck of ZFNs and launching second generation designer nucleases before RNA-guided systems emerged.

Ref: Mak et al. Science 2012 TALE DNA crystal; Moscou Bogdanove Science 2009 RVD code DNA bases.

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Transgenic Plant -2

TALENs are derived from proteins of:

Transcription activator-like effector nucleases originate from plant pathogenic bacteria Xanthomonas genus comprising Xanthomonas oryzae pv. oryzae causing bacterial blight of rice and Xanthomonas citri causing citrus canker. Pathogen delivers effector proteins via type III secretion apparatus into plant cell cytoplasm mimicking eukaryotic transcription factors: N-terminal type III secretion signal, central repeat domain 12-30 tandem repeats of 33-35 amino acids differing at two positions conferring DNA specificity, C-terminal activation domain and nuclear localization signals. Once in nucleus, TALE binds promoter of susceptibility gene such as SWEET sucrose efflux transporters OsSWEET11,14 inducing expression to feed bacteria. Boch and Bonas 2009 cracked code linking RVD to bases. TAL effector scaffold repurposed by replacing activation domain with FokI nuclease catalytic domain creating TALEN able to cut at designer loci. Rice edited to mutate SWEET promoter EBE element preventing Xanthomonas activation while preserving endogenous function achieved bacterial blight resistance demonstrating direct application of pathogen biology to crop improvement before CRISPR adoption.

Ref: Boch Science 2009 326:1509 TALE Xanthomonas; Bogdanove Curr Opin Microbiol; Moscou Bogdanove 2009.

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Transgenic Plant -2

Repair of ZFN-induced breaks commonly occurs via:

Repair of nuclease induced breaks determines editing outcome and is governed by competitive engagement of canonical non-homologous end joining NHEJ and homology-directed repair HDR pathways. NHEJ active throughout cell cycle especially G1, initiated by Ku binding, DNA-PKcs activation, end processing by Artemis and polymerase mu, ligation by Lig4-XRCC4-XLF complex. It is error-prone with indels up to tens of base pairs at cleavage site leading to gene disruption valuable for knockout breeding. Alternative microhomology mediated end joining using Pol theta also produces deletions. HDR pathway requires extensive 5 to 3 end resection by CtIP-MRN generating 3' single-stranded overhangs coated by RPA then replaced by Rad51 filament facilitating homology search, strand invasion into sister chromatid or exogenous donor template, DNA synthesis using homologous sequence, resolution via synthesis dependent strand annealing. HDR restricted to S and G2 phases when sister chromatid available, efficiency generally less than NHEJ. Strategies to boost HDR include supplying single-stranded oligodeoxynucleotide donor with 50-90 bp arms, synchronizing cells, inhibiting NHEJ with SCR7 Lig4 inhibitor or i53 blocking 53BP1, or using Cas9 nickases favoring HDR. Understanding balance critical for trait introgression.

Ref: Ceccaldi et al. Nat Rev Genet 2016 17:147 NHEJ HDR; Symington Lenski Annu Rev Genet; Alberts.

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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

FokI nuclease requires __________ for DNA cleavage.

FokI restriction enzyme from Flavobacterium okeanokoites is Type IIS enzyme recognizing GGATG and cutting 9/13 downstream, separated into N-terminal DNA recognition domain and C-terminal cleavage domain. Nuclease domain isolated for genome editing displays catalytic motif PD...D/EXK coordinating Mg2+ for phosphodiester hydrolysis. Critical feature discovered by crystallography is cleavage domain inactive as monomer because catalytic center requires dimerization to align two active sites across DNA duplex forming DSB. Biochemical assays show monomeric FokI binds but cannot incise even with Mg2+. In ZFN context, two monomers must bind in close proximity with spacer length 5-7 bp optimal allowing C-terminal domains to interact via hydrophobic interface. Dimerization requirement provides double checkpoint enhancing specificity, since single ZFN binding does not lead to breakage unlike monomeric nucleases. Engineering obligate heterodimer FokI variants DD: ELAN and RR:DRSH introducing electrostatic repulsion for homodimers further reduces off-target activity, improving therapeutic window. Same principle exploited in TALEN and dimeric Cas9-FokI systems where spatial proximity essential for catalysis ensuring genome stability.

Ref: Bitinaite et al. PNAS 1998 95:10570 FokI dimer; Miller Nat Biotech 2007 Heterodimer; Waugh 1993.

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Transgenic Plant -2

Each zinc finger domain recognizes:

Specificity principle of C2H2 zinc finger modular recognition lies in each finger contacting three base pairs. Structural biology of Zif268-DNA complex by Pavletich and Pabo 1991 revealed that alpha helix of each finger inserts into major groove, amino acid residues at positions -1, 2, 3 and 6 relative to helix start make sequence-specific contacts. Triplet code allows prediction: for example Arg at -1 contacts guanine, Asp at 2 contacts cytosine, pattern learned from SELEX. Therefore three finger protein recognizes 9 bp, four finger 12 bp, six finger 18 bp. Context dependence exists because neighboring fingers influence docking, sometimes overlapping fourth base cross-strand contact extending recognition beyond three bases, but canonical model simplifies design. To achieve unique genomic address in rice genome ~430 Mb, 12 bp occurs ~250 times, 18 bp statistically once, but paired ZFNs double site to 18-36 bp effectively unique plus dimer requirement increases specificity reducing off-target cleavage versus restriction enzymes recognizing 6 bp. Modular assembly libraries of 64 triplet-specific fingers enable construction of ZFN targeting almost any GNNGNNGNN sequence, though AT-rich sequences remain challenging requiring selection approaches.

Ref: Pavletich Pabo Science 1991 252:809 Zif finger 3 bp; Wolfe Ann Rev Biophys 2000; Beerli 2002.

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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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