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ZFN, TALEN, and CRISPR/Cas-based methods for genome engineer

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Anna Weber

on 11 February 2014

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Transcript of ZFN, TALEN, and CRISPR/Cas-based methods for genome engineer

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
How do we introduce them?
microinjection


transfection via electroporation


transfection via cationic lipid-based reagents

nucleases encoded on:
viral vectors
plasmid DNA
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!

traditional approaches:
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
nucleases
transcriptional effectors
recombinases
transposases
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
TALEs
transcription activator-like effectors
naturally occuring in a plant pathogenic bacteria Xanthomonas
N-terminal:
nuclear localization signal
central domain:
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)
C-terminal:
numerous effector domains
e.g. Fok1 endonuclease
How does editing with nucleases work?
nucleases induce double-strand break (DSB)
homology-directed repair
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
non-homologous end-joining
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
reverse genetics
gene inactivation via homologous recombination:
+ precise genetic manipulations
- low efficiency, time-consuming and with potential for mutagenic effects
RNAi:
+ 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
ineffective

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
commercially available
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
CRISPR-locus:
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
RNA-guided
naturally in bacteria to provide immunity against invading foreign DNA
Comparison of ZFN, TALEN and CRISPR
ZFN
TALEN
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
off-target effects?
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
therapeutic applications
outlook
correction of underlying cause of disease
insertion of therapeutic transgenes
DNA binding domain - construction
specificity
off-target effects and toxicity
delivering methods
therapeutically relevant?
homologous repair vs. non-homologous end joining
TALEs
construction of tandem repeats
Golden Gate Cloning
--> standardized, multi-part DNA assembly

solid-phase based TALE repeats assembly methods:
FLASH
ICA

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
commercially available
PAM necessary in order for Cas9 to cleave
not (yet) commercially available
off-target effects? toxicity?
other opened questions
NLS
NLS
Full transcript