Topic 10 - Gene editing

Gene editing

changing nt sequences (insert/deletion/sub), at specific location,use DNA endonuclease/ dsDNA break

DNA endonuclease

cleave DNA, specific location, result in dsDNA break

Double stranded DNA break (DSB)

allow for gene to be modified, naturally (exposure extrernal/internal agents) or artifically, repair by NHEJ or HDR

Non homologous end joining

prone to error/nt lost, occur before replication (G1), allows for gene Knockouts

Non homologous end joining steps

Proteins (PKcs, Ku70, Ku80) recognized dsDNA break, trim nt to make blunt end, DNA ligase glues back together

Homology directed repair

use DNA template, after DNA replication (G2), allows for gene insertion/modification

Homology directed repair steps

dsDNA break in one chromatid, Nuclease trim off portion of broken, Rad51 bind undamaged chromatid, Strand invasion forms displacement loop, DNA replicates using sister chromatid as template, Blunt end ligated by DNA ligase

Gene editing tools

exploit natural DNA repair mechanism, sequence specific DNA binding domian (ZFN and TALEN), complex with RNA molecule (CRISPR/Cas9)

Zinc Finger Nuclease (ZFN)

from African claw frog, DNA binding loop with zinc structures, target human and stem cells, use endonuclease Fokl, cleave by dimerise (pinch DNA), Costly and difficult to make, off target cleavage (not as specific)

Transcription activator-like effector nuclease (TALENs)

from bacteria Xanthomonas, tandem array of DNA binding repeats, target model organisms, use endonuclease Fokl, cleave by dimerise (pinch DNA), Costly to make, off target cleavage (not as specific)

CRISPR/Cas9

in prokaryotes, DNA nuclease in genome beside (CRISPER associate (cas) genes), natural defense mechansim

CRISPR/Cas9 defense mechanism steps

CRISPR transcribed into ncRNA, tracer gene transcribed into tracrRNA, Cas9 is produced, processed into crRNA by cas encoded RNases, TracrRNA/crRNA/Cas complex cleaves foreign DNA (deactivates), cleaved DNA stored in CRISPR region of genome (passed to progeny)

CRISPR gene editing steps

target any sequence, crRNA and tracerRNA fused to single guide RNA (sgRNA), insert target DNA (unique crRNA), Cas9 used as endonuclease to make dsDNA break, NHEJ used for deletions/insertions (in genome) or HDR use supplied DNA for synthesis

Cas9

endonuclease, HNH domain - cleaves complementary strand to crRNA, RuvC domain - cleace opposite strand, PAM - protospacer/ destabilies sequence for crRNA pairing, class 2 type 2

Class 1 Cas

Multi subunit, type 1/3/4

Class 2 Cas

single subunit, type 2/5/6, Cas 9 and 12 target DNA, Cas 13 targets RNA

How recombinant in DNA/CRISPR into cells

transformed into plasmid and inserted into Agrobacterium, coculture or injected in plant tissue, or plasmid coated in gold and plant tissue bombarded with particles, plant grwon to observe phenotype

Agrobacterium

natural alter host genome, plant pathogen, causes uncontrolled cell division - tumour inducing plasmid (Ti), plasmid contain transfer DNA (T-DNA) that recombines into host

Agrobacterium DNA insertion

T-DNA trancribed, virulence translated, conjugative transfer proteins made, T-DNA insert randomly into genome

Agrobacterium mediated transformation

Ti plasmid disarmed (remove opine/tumor genes), gene of intertest added in T-DNA border (transformation vector), both plasmid back into agrobacterium

Application gene editing

plant research, economically important plants, mostly NHEJ repair for knockout to determine gene function

Genetically modified organsims (GMOs)

organsimhad their DNA changed by artificial means, allowed in some countries if could be achieved by selective breeding (no foreign DNA)

CRISPR/CAS9 ownership

Doudna and Charpentir filed for patent first but Zhang paid for his patent to be expedited, current ongoing court case by Zhang maintains patent, Doudna and Charpentir won 2018 Nobel prize for tho