Transcript Document

Zinc Finger Engineering
Fancy Fingers in Gene Repair: Human Genome Engineering
D. Sundar
Centre for Bioinformatics, Pondicherry University
Presentation Outline
• Zinc Finger Proteins
• Zinc Finger Platform Technology
• Applications
What is a Zinc Finger?
- Naturally occurring class of DNA
transcription factors
- Originally recognized in the
transcription factor TFIIIA.
- Structure
• 24-30 amino acids long
• Consists of a simple ßß-fold
(two anti-parallel beta strands
and an alpha-helix)
• Cys2His2: Two cysteine and two
histidine ligands bind a zinc ion
Why Zinc Finger Proteins?
§
C2H2 zinc finger DNA-binding proteins have
proven to be most versatile.
§
C2H2 zinc fingers are found in 2% of all human
genes and are the most abundant class of
DNA-binding domains found in human
transcription factors.
§
Their structure makes them an ideal framework
for engineering to bind to selected target
sequences.
Why Zinc Finger Proteins?
(Contd…)
§
Each finger recognizes and binds to three base
pair sequence of DNA
§
Three such fingers can be joined together to
bind a 9-base pair sequence and
correspondingly 6 fingers an 18 base pair
sequence.
How can we use zinc fingers to recognize
long stretches of DNA ?
Linker
By stringing several of these zinc fingers in tandem,
we can create multiple zinc finger protein
that can bind to any sequence of interest
THE ZINC FINGER ADVANTAGE !!
Sundar et. al.: Nuc. Acids Research. (2005)
Key base contacts in the Zif-DNA complex
Target site
overlap
Finger 1
Finger 2
Finger 3
Kim & Berg: Nat. Struc. Biol: 3, 940-45, 1996
Advances in Zinc finger engineering
§
One of the most important and successful
strategies for selecting zinc fingers with
affinity for desired target site have been
Phage Display.
§
Few other alternative systems for zinc finger
selection have been developed:
§
§
§
Yeast One-hybrid System (Bartsevich & Juliano, 2000)
Mammalian One-hybrid System (Blancafort et al., 2003)
Bacterial Two-hybrid System (Joung et al., 2000)
Phage display
Method of generating billions of protein variants and
selecting for those bind to a particular target
A protein is fused to a viral coat protein of the phage
The virus is allowed to reproduce in culture, where it
copiously makes new copies of itself
The phage virus displays these proteins on the surface
of the virions,
Selection is done in vitro by simply passing the viral stew
over a stationary phase containing the target substrate.
Those that can bind well, and the ones that bind the best
will bind the tightest
Alternative Systems
 The three alternative strategies reported so far have
the advantage that zinc fingers can be identified in a
single round (instead of multiple rounds for phagedisplay)
• The Bacterial two-hybrid system, in particular, has the
advantage that very large libraries can be constructed
and evaluated.
Basal transcription by RNA polymerase
(Catalyzes the synthesis of RNA directed by DNA as a template = transcription)
a
a
b
b'

-63
-35
-10
Pwk
Reporter
a : assembly and binds to UP
b+b’ : form catalytic center
 : binds -10 and -35 of promoter to confer
specificity during initiation
Selection System
A bacterial,one-hybrid genetic system for
evaluating and evolving zinc finger affinity
and specificity for DNA.
Initial design of one-hybrid system
+
Co-transformed into
E.coli cells (DH5aE)
Fusion plasmid (FP)
Reporter plasmid (RP)
Selected for activity on increasing concentrations of
chloromphenicol plates or for GFP fluorescence levels
Initial Data
Initial Data for Antibiotic System
150
125
Highest Conc 100
of Cm on which
75
colonies grew
(microgram/ml) 50
25
0
FP(without
ZiF)
ZiF)
RP
(target
siteFP(with
@ -63)
RP(Target site @ -63)
Initial Data for Fluorescence System
RP(Target site @ -62)
FP(without ZiF) Geo Mean = 1.54
FP (with ZiF) Geo Mean = 1.96
Arbitrary Fluorescent Units
Construction of Incremental Truncation
Libraries to Improve System
Fusion plasmid (FP)
Reporter plasmid (RP)
To create linkers ranging
from 4 to 23 amino acids
To center the QNK-binding
site at locations varying
from -81 to -30
“Linker Library”
“Binding-site Library”
Creation of library
• We wanted to create a library containing every one base
pair deletion of a gene fragment.
• Combinatorial approach
• Incremental Truncation
Incremental Truncation
Open up the plasmid by double digestion
Exo III nuclease treatment
asdasd
Salt concentration (Nacl)
Time-dependent sampling !!
Ostermeier, Nixon & Benkovic: Proc. Natl Acad. Sci :96, 3562-67, 1999
Mung bean nuclease treatment
susceptible
resistant
Klenow treatment
asdasd
Diverse Library with every one base deletion
Selection of library members
Linker Library
Binding Site Library
+
+
Select on increasing
levels of Cm
Sort, using a flow
cytometer, based on
total cell fluorescence
(@ 530nm)
A closer target site and a longer linker
yields the highest transcription
Optim ized Antibiotic System
700
600
Minimum
500
Inhibitory
Concentration
400
(MIC)
(microgram/ml 300
Cm) when
expressing the 200
protein
100
N/D
0
RP(Target site @-63)
FP(w ithout ZiF)
FP(w ith ZiF)
RP(Target site @ -55)
FP(w ith Zif and 22 a.a. linker)
A closer target site and a longer linker
yields the highest transcription
RP (target site @ -62)
FP(without ZiF) Geo Mean = 1.44
FP (with ZiF and 22 a.a. linker) Geo Mean = 2.34
Arbitrary Fluorescent Units
RP (target site @ -55)
FP(without ZiF) Geo Mean = 1.81
FP (with ZiF and 22 a.a. linker) Geo Mean = 13.64
Arbitrary Fluorescent Units
Zinc Finger Nucleases
• Chimeric nucleases are novel restriction enzymes
with tailor made sequence specificities.
• They have Zinc fingers as DNA binding domain(N
terminal) fused to FokI cleavage domain (C
terminal) by a (G4S)3 linker.
Dimerization of the cleavage domain
1. Dimerization of the nuclease domain is required for DNA
cleavage.
2. Inverted repeats are preferred substrates for Zinc finger
Chimeric nucleases.
Bibikova et al: Mol Cell Biol 2001 Jan;21(1):289-97
Zinc Finger Nucleases
Zinc Fingers in action !!
• Towards conferring immunity to HIV-1
infection
Estimate of AIDS spread by 2010
Presently 42 million are living with HIV.
22 million are already killed by the virus.
National Intelligence Council predicts that by 2010 there
will be between 50 million and 75 million cases in India,
China, Russia, Ethiopia and Nigeria.
Washington Post, October 1, 2002.
18 to 26 percent of adult population will be infected in
Ethiopia and Nigeria and 4 to 5 percent will be infected
in India.
HIV
HIV is a retrovirus that infects CD4+ T
cells, Macrophages, Dendritic cells etc.
It carries two RNA copies of its genome
and viral Reverse Transcriptase and
accessory proteins.
Virus gains entry into cells by attaching
to two receptors, the major receptor
CD4 and a Chemokine co-receptor.
The RNA is reverse transcribed and
integrated to the host genome.
CCR5 Δ32 mutation confers
resistance to HIV infection
Subjects with homozygous 32bp deletion in CCR5 gene
remain uninfected despite extensive exposure to HIV-1.
Heterozygous subjects show decreased efficiency of HIV-1
entry and replication in CD4+ T-cells and delay in the
progression of the disease. (Landau et al.1996).
Homozygous mutation is present in 1% of Caucasians and
is rare among Asians and Africans
Heterozygous mutation is present in about 10% of
Caucasians.
No deleterious effect has been detected due to the mutation.
CCR5 Δ32 mutation
The 32 bp deletion causes a frame shift mutation in
the CCR5 gene corresponding to the second extra
cellular loop of the receptor, generating a stop
codon in the TMD 5, encoding a severely truncated
molecule which fails to reach the cell surface.
The wild type receptor protein is 352 AA. The
mutated receptor protein is 215 AA.
Various approaches to reduce CCR5 expression
.
Down regulation of CCR5 expression by targeting
multiple cleavage sites in the CCR5 mRNA using
anti-CCR5 heterotrimer ribozymes (Bai et al. 2001)
SiRNA against the CCR5 gene expression. (Lee et
al. unpublished)
Functional deletion of CCR5 receptors by
intracellular immunization (Barbas et al. 2000)
Hypothesis
Functional deletion of the CCR5 receptor can be
achieved by targeted mutagenesis of the CCR5 gene
using Chimeric Nuclease technology, thereby, conferring
resistance to HIV-1 infection.
Using this technology in CD34+ Stem cells, it might be
possible to repopulate the host with HIV-1 resistant CD4+
cells.
Isolate patient’s
CD34+ cells
Mutate CCR5
gene using
chimeric
nucleases
Select cells
with CCR5
mutation
Repopulate patient’s
with mutated CCR5
stem cells
Experiment design and methods
1. Identification of specific target sites within the
CCR5 gene.
2. Design zinc finger proteins that bind to the target
sequences.
3. Convert zinc finger proteins into chimeric
nucleases.
4. Deliver the chimeric nucleases into CD34+
stem/progenitor cells.
5. Identify CD34+ cells that are resistant to HIV-1
infection.
6. Monitor these cells for functional deletion of
CCR5.
Identification of a specific target site
421 tgaagagcat gactgacatc tacctgctca acctggccat ctctgacctg tttttccttc
481 ttactgtccc cttctgggct cactatgctg ccgcccagtg ggactttgga aatacaatgt
541 gtcaactctt gacagggctc tattttatag gcttcttctc tggaatcttc ttcatcatcc
601 tcctgacaat cgataggtac ctggctgtcg tccatgctgt gtttgcttta aaagccagga
661 cggtcacctt tggggtggtg acaagtgtga tcacttgggt ggtggctgtg tttgcgtctc
721 tcccaggaat catctttacc agatctcaaa aagaaggtct tcattacacc tgcagctctc
781 attttccata cagtcagtat caattctgga agaatttcca gacattaaag atagtcatc
841 tggggctggt cctgccgctg cttgtcatgg tcatctgcta ctcgggaatc ctaaaaactc
901 tgcttcggtg tcgaaatgag aagaagaggc acagggctgt gaggcttatc ttcaccatca
961 tgattgttta ttttctcttc tgggctccct acaacattgt ccttctcctg aacaccttcc
1021 aggaattctt tggcctgaat aattgcagta gctctaacag gttggaccaa gctatgcagg
1081 tgacagagac tcttgggatg acgcactgct gcatcaaccc catcatctat gcctttgtcg
1141 gggagaagtt cagaaactac ctcttagtct tcttccaaaa gcacattgcc aaacgcttct
NCBI: Homo sapiens Chem R13(X91492)
Chimeric nuclease bound to CCR5 target site
(G4S)3
Zif
3
2
1
FN
5’-GTC CCC TTC ctggctcactat GCT GCC GCC-3’
3’-CAG GGG AAG gaccgagtgata CGA CGG CGG-5’
5
4
6
FN
Zif
(G4S)3
5’-GTC CCC TTC ctggct cactat GCT GCC GCC-3’
3’-CAG GGG AAG gaccga gtgata CGA CGG CGG-5’
Program to identify ZF target sites
§
We are developing bioinformatic tools :
§ To identify key targets for ZF for the designs that
we already have.
§ To identify inverted targets sites for our ZFN to
bind and make a DSB.
§ Developing a Database of all documented ZF so
far. This also has provisions for appending newly
evolved ZF proteins that we are going to solve
using our selection method.
Summary
• Gene editing of all of the genes encoded in
human cells will become possible.
• Highly efficient and directed site-specific
modification of the plant and animal
genome without selection to make
transgenics will be feasible.
Questions ?