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

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 activati

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 rear

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-stra

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

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

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

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 re

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 introduci

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+

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 n

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 r

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

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