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ZFN, TALEN, and CRISPR/Cas-based methods for genome engineer
Transcript of ZFN, TALEN, and CRISPR/Cas-based methods for genome engineer
How do we introduce them?
transfection via electroporation
transfection via cationic lipid-based reagents
nucleases encoded on:
in vitro: mRNA
Genome engineering - why is this an important topic?
More and more genetic sequences are available, but you need to link genotype to phenotype!
What do these proteins look like?
Tools for genome engineering:
1) target specific DNA binding domain (programmable)
2) DNA cleavage domain
...or other effector domain
DNA & histone methyltransferases
chromatin remodeling factors
ZFN = Zinc-finger nucleases
use of unnatural arrays with more than 3 zinc-finger domains
construction of synthetic proteins possible
new proteins recognize up to 18 bp --> specificity within genome!
a target site consists of two zinc-finger binding sites seperated by a 5-7 bp spacer sequence where the cleavage domain binds
transcription activator-like effectors
naturally occuring in a plant pathogenic bacteria Xanthomonas
nuclear localization signal
DNA binding domain
--> consists of repeats of 33- 35 amino acids, each repeat recognizes 1 base pair
--> determination of specificity by two hypervariable amino acids (RVD's = repeat variable di-residues)
numerous effector domains
e.g. Fok1 endonuclease
How does editing with nucleases work?
nucleases induce double-strand break (DSB)
DSB increases chance of homology-directed repair
--> a provided plasmid with homology arms can induce integration of transgenes
--> induce mutations, insertions or deletion through linear donor sequences (<50bp homology) or single-strand oligonucleotides
Error-prone, but very fast and common repair system
often gene knock-out through small deletions/inserts (frame-shift)
deletions, inversions and translocations of large chromosomal fragments possible
introduction of large transgenes
CRISPR = clustered regulatory interspaced short palindromic repeat
gene inactivation via homologous recombination:
+ precise genetic manipulations
- low efficiency, time-consuming and with potential for mutagenic effects
+ rapid, inexpensive, high-throughput,
- knock-down is incomplete, unpredictable off-target effects, inhibition only temporary
forward genetic screens
chemical mutagenesis, transposon-mediated mutagenesis
randomly induced mutations
surface amino acids on α-helix contact typically 3 bp in major groove of DNA
highly conserved linker sequence between several zinc finger domains
"modular assembly": you choose the zinc-fingers targeting your triplets from a preselected library
selection-based-approaches: you select arrays from randomized libraries
How do we construct selective ZFNs?
How can we use Zinc finger domains for our purpose?
What does a Zinc finger domain look like?
engineered single RNA chimera of crRNA and tracrRNA (named guiding RNA, gRNA) can be used to direct Cas9
a target sequence needs to be preceded by NGG, because that sequence always precedes a spacer
short direct repeats (21-47 bp)
spacers = segments of foreign DNA
surrounded by Cas genes (CRISPR-associated genes)
spacers are transcribed and processed into crRNA
crRNA forms a double strand with tracrRNA (trans-activating RNA), this double strand leads Cas9 nuclease to DNA sequence complementary to crRNA
naturally in bacteria to provide immunity against invading foreign DNA
Comparison of ZFN, TALEN and CRISPR
zinc finger domains for almost all triplets
already successfully used to correct disease related mutations in many cases
can be injected as a purified protein
combinable with other proteins
cloning of repeats is technically challenging
large size of TALEs limits the delivery in certain viral vector plasmids
targeting efficiency cannot be predicted reliably
sequence has to start with T base
precise cleavage, high efficiency
already successful in mouse and human cell lines, human pluripotent stem cells, zebrafish
easy cloning strategy --> only RNA molecule needs to be synthesized
Cas9 can be converted into a nickase
multiple gRNAs can be used to modify multiple sites simultaneously
correction of underlying cause of disease
insertion of therapeutic transgenes
DNA binding domain - construction
off-target effects and toxicity
homologous repair vs. non-homologous end joining
construction of tandem repeats
Golden Gate Cloning
--> standardized, multi-part DNA assembly
solid-phase based TALE repeats assembly methods:
large number of TALENs can be constructed fast
context effects, different specificity
re-engineering of linkage necessary
ZFN production costly, laborious
not all corresponding ZFs discovered
combinable with other proteins
great design flexibility
methods to avoid cloning problem (FLASH, ICA, Golden Gate)
well established method
PAM necessary in order for Cas9 to cleave
not (yet) commercially available
off-target effects? toxicity?
other opened questions