Zinc Finger Engineering

Fancy Fingers in Gene Repair: Human Genome Engineering

D. SundarCentre for Bioinformatics, Pondicherry University

Presentation Outline

• Zinc Finger Proteins• Zinc Finger Platform Technology• Applications

- 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

What is a Zinc Finger?

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 ?

By stringing several of these zinc fingers in tandem, we can create multiple zinc finger protein that can bind to any sequence of interest

Linker

Sundar et. al.: Nuc. Acids Research. (2005)

THE ZINC FINGER ADVANTAGE !!

Key base contacts in the Zif-DNA complex

Finger 1 Finger 2 Finger 3

Target site overlap

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 displayMethod 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

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

The phage virus displays these proteins on the surface of the virions,

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 phage-display)

• The Bacterial two-hybrid system, in particular, has the advantage that very large libraries can be constructed and evaluated.

Alternative Systems

: assembly and binds to UP’ : form catalytic center

: binds -10 and -35 of promoter to confer specificity during initiation

Basal transcription by RNA polymerase

-35 -10

'

-63 Pwk Reporter

(Catalyzes the synthesis of RNA directed by DNA as a template = transcription)

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

Reporter plasmid (RP)Fusion plasmid (FP)

+Co-transformed into E.coli cells (DH5E)

Selected for activity on increasing concentrations of chloromphenicol plates or for GFP fluorescence levels

Initial Data

RP(Target site @ -62)FP(without ZiF) Geo Mean = 1.54

FP (with ZiF) Geo Mean = 1.96

RP (target site @ -63)

Initial Data for Antibiotic System

0

25

50

75

100

125

150

FP(without ZiF) FP(with ZiF)

Highest Conc of Cm on which colonies grew

(microgram/ml)

RP(Target site @ -63)

Initial Data for Fluorescence System

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 acidsTo 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

asdasd

Incremental Truncation

Ostermeier, Nixon & Benkovic: Proc. Natl Acad. Sci :96, 3562-67, 1999

Exo III nuclease treatment

Salt concentration (Nacl)

Time-dependent sampling !!

Open up the plasmid by double digestion

asdasd

susceptibleresistant

Mung bean nuclease treatment

Klenow treatment

Diverse Library with every one base deletion

Selection of library members

+ +

Select on increasing levels of Cm

Linker Library Binding Site Library

Sort, using a flow cytometer, based on total cell fluorescence (@ 530nm)

A closer target site and a longer linker yields the highest transcription

Optimized Antibiotic System

0

100

200

300

400

500

600

700

RP(Target site @-63) RP(Target site @ -55)

Minimum Inhibitory

Concentration (MIC)

(microgram/ml Cm) when

expressing the protein

FP(without ZiF) FP(with ZiF) FP(with Zif and 22 a.a. linker)

N/D

A closer target site and a longer linker yields the highest transcription

RP (target site @ -55)

FP(without ZiF) Geo Mean = 1.81

FP (with ZiF and 22 a.a. linker) Geo Mean = 13.64

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

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.

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

Dimerization of the cleavage domain

Zinc Finger Nucleases

• Towards conferring immunity to HIV-1 Towards conferring immunity to HIV-1 infectioninfection

Zinc Fingers in action !!

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.

18 to 26 percent of adult population will be infected in Ethiopia and Nigeria and 4 to 5 percent will be infected in India.

Washington Post, October 1, 2002.

Estimate of AIDS spread by 2010

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.

HIV

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 confers resistance to HIV infection

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.

CCR5 Δ32 mutation

. 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)

Various approaches to reduce CCR5 expression

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

Select cells with CCR5 mutation

Repopulate patient’s with mutated CCR5 stem cells

Mutate CCR5 gene using

chimeric nucleases

Hypothesis

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.

Experiment design and methods

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)

Identification of a specific target site

5’-GTC CCC TTC ctggctcactat GCT GCC GCC-3’

3’-CAG GGG AAG gaccgagtgata CGA CGG CGG-5’

3 2 1FN

Zif(G4S)3

64 FN

Zif (G4S)3

5

5’-GTC CCC TTC ctggct cactat GCT GCC GCC-3’

3’-CAG GGG AAG gaccga gtgata CGA CGG CGG-5’

Chimeric nuclease bound to CCR5 target site

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 ?