Precision Genome Editing in Mammalian Cells Using Engineered Zinc Finger Proteins

Greg Davis1* and Trevor N Collingwood

Sangamo BioSciences, Inc., 501 Canal Blvd, Ste. A100, Richmond, California 94804, USA.1 Sigma-Aldrich Biotechnology, 2909 Laclede Ave, St. Louis, MO 63103, USA

Introduction Western blot Normal Homology WT Locus ZFN-Driven Gene

ZFNs Drive Efficient Targeted Integration in Stem Cells
1F 2B DG44 B3 WT Directed Repair Insertion
In this instance targeted insertion was
Rational genome engineering in mammalian cells has enormous potential across basic research, drug- Donor only ZFNs + Donor
GS Randomly induced DSB ZFN-induced DSB performed in Human Mesenchymal Stem
discovery and cell-based medicines. Existing methods for targeted gene knock out or site-specific
Cells. The target locus was the IL2Rγ
gene insertion rely on homologous recombination, which occurs at the low frequency of ~ 1 event in
Sister chromatid used Homologous donor plasmid gene. The homologous Donor contained a
10e6 in most somatic cells. The scale of screening effort and the time required to isolate the targeted event DHFR as repair template carrying transgene for use
as repair template promoter-driven GFP cDNA.
can therefore be prohibitive. Thus, there exists a strong need for a technology that can rapidly achieve
genomic editing with high speed and efficiency and so greatly reduce overall engineering effort. Beta -Tubulin Locus repaired with Transgene inserted
Cells were transfected with either the Donor
WT sequence
into Locus alone, or with Donor plus ZFNs specific
We have developed a novel approach that enables high-frequency genome editing via the application of
for the target locus. The transfected cells
designed zinc finger protein nucleases (ZFNs). We demonstrate here the application of ZFNs to both targeted Targeted Insertion of a GFP cDNA Near an Endogenous Promoter
ZFNs were grown for a further 1 month (without
gene knockout and targeted gene insertion. /-
GS DHFR -/- clone 1F (PPP1R12C) antibiotic selection) to allow the non-
1.0E+06
integrated GFP donor plasmid to disappear
Engineered Zinc Finger Protein Nucleases (ZFNs) 8.0E+05
from the cells.

Total Cells
6.0E+05 (includes splice acceptor
Engineered zinc finger proteins (ZFPs) can be designed Growth of GS -/- clone B3 + 2A linker) Significant GFP fluorescence was only detected
4.0E+05
to bind virtually any specified DNA sequence. ZFN -R
-R 6.0E+05
in cells transfected with Donor and ZFNs.
5.0E+05 +HT +Glu 2.0E+05
ZFPs are be coupled with the catalytic domain of the +HT - +ZFNs

Total Cells
4.0E+05 0.0E+00
restriction endonuclease FokI to create a Zinc Finger - +Glu 0 1 2 3 4 5

Nuclease (ZFN). 3.0E+05 - - Days in culture Advantages of ZFN-Mediated

When a pair of ZFNs bind to their target site at
2.0E+05
Targeted Gene Insertion
1.0E+05 ZFNs cleave the endogenous site in intron 1 of the target gene. The homologous donor contains a
an endogenous genomic locus a catalytically active ZFN -L
ZFN-L 0.0E+00
GS -/- DHFR -/- clone 2B promoterless GFP cDNA linked to a splice acceptor (SA), flanked by sequence homologous to the target locus.  Single step targeted insertion into native loci
1.6E+06
FokI dimer forms. The DNA is thus cleaved to create a 0 1 2 3 4 5
Days in culture
1.4E+06 During ZFN-induced HDR the GFP is inserted at the cleavage site so that its expression becomes driven by the  No need to pre-insert a “genomic landing pad”
double strand break (DSB). 1.2E+06

Total Cells
1.0E+06 endogenous promoter.
The DSB is then repaired by either or two natural mechanisms that determine the nature of the genetic 8.0E+05
 Target identical sites in all cells of same species
6.0E+05
change at the target locus  Monoallelic or biallelic insertion
4.0E+05
2.0E+05
Targeted Insertion of the GFP is ZFN Dependent
• Non-homologous end joining (NHEJ): we use this for gene disruption 0.0E+00
 High efficiency means:
• Homology dependent repair (HDR): we use this for gene insertion
0 1 2 3
Days in culture
4 5 PCR FACS • Minimal screening effort
Three human cell lines were D D+Z Donor only Donor + ZFNs
transfected with the GFP • Antibiotic selection is not necessary
ZFN-Mediated Targeted Gene Disruption Targeting FUT8 in a GS-/- DHFR-/- Cell Line Creates Donor only (D) or Donor + 0.78% 13.1%
a GS-/- DHFR-/- FUT8-/- Triple Knockout ZFNs (D+Z).
Summary
1. Wild type cells
GS-/- DHFR-/- cells were treated with PCR amplification of the
ZFNs targeting the FUT8 gene in the target locus shows a Using site-specific zinc finger protein nucleases (ZFNs), we can now routinely achieve biallelic targeted gene
Transient transfection of ZFN plasmids to
2. introduce a site-specific double stranded critical Fut motif II (exon 10) specific integration event disruption at frequencies of 1-5%. Moreover, we attain these levels of performance without the use of
DNA break in the target gene.
3 days x only in the presence of antibiotic selection. Thus, it now becomes feasible to disrupt multiple genes in a single cell. To that end,
Following dilution cloning, 4/200
Donor + ZFNs. we demonstrate here the effectiveness of the ZFN approach in generating single, double and triple gene
Break repaired imperfectly by non- clones (2%) showed biallelic
3. homologous end-joining (NHEJ). knockouts in a mammalian cell line.
This destroys gene function. disruption of FUT8 FACS analysis also shows a 0% 3.7%
ZFN-dependent increase in We also show that ZFNs can drive single step targeted integration into native endogenous loci at frequencies
1 month
4. x Gene disrupted The inability of clones to bind Fut motif I
GFP fluorescence. >10%. This too is achieved without the need for antibiotic selection. In this study and in recently published
fluorescein-labeled Lens culinaris Fut motif II
5. Single cell clone Fut motif III ……... WT, no FLCA Both assay indicate a work1,2, such results have been achieved in a variety of cell types, including human embryonic and
agglutinin (FLCA) confirms FUT8-/- _____ WT, + FLCA
Site of ZFN Cleavage targeted integration mesenchymal stem cells, CD34+ cells, and several different transformed cell lines.
phenotype _____ 14C1, + FLCA (+1/+4)
_____ 35F2, + FLCA ( Δ4/ Δ5) frequency of up to 13%. Zinc Finger Nuclease technology thus offers an important advance in cell engineering, with the potential to
Glutamine Synthetase (GS) Knockout in CHO Cells
No antibiotic significantly impact both academic research and industrial cell-based applications.
CHO-S cells grown in suspension were treated with a pair of ZFNs that target exon 6 of GS
Advantages of ZFN-Mediated selection was used. 0.65% 1.7% 1. Moehle et al., 2007, PNAS 104, 3055.
The treated pool was cloned by limiting dilution without selection 2. Lombardo et al., 2007,Nat. Biotech. 25, 1298.
GeneKnockout Technology
Genetic analysis (PCR / sequencing) of 54 clones showed that 10 clones (19%) had both copies of the
GS gene disrupted  Rapid and permanent targeted disruption of (any) endogenous locus
Contact Details
Most mutations were frame shifts or large deletions that resulted in (i) the complete loss of GS protein  Monoallelic or biallelic gene disruption
expression; (ii) no growth in the absence of L-glutamine Sangamo BioSciences – Sigma-Aldrich For enquiries regarding ZFN technology please contact:
 ZFNs are transferable within and (often) across species
Greg Davis, PhD. Trevor Collingwood, PhD.
Announced July 10th, 2007
 ZFNs are expressed transiently Sigma-Aldrich Sangamo BioSciences
Genotype of clones Western blot of clones Sorting the Cells by GFP Fluorescence Enriches Collaboration agreement to provide researchers Missouri, USA California, USA
 Minimal screening effort (few hundred clones)
for the Targeted Insertion Event with broader access to ZFP / ZFN technology Phone: (314) 771-5765 Phone: (510) 970-7805
 No selection required greg.davis@sial.com tcollingwood@sangamo.com

 Multigene targeting (trait stacking) readily achievable

 Enables late-stage re-engineering of important cell lines e.g. recombinant protein high producers
Acknowledgements
Cell Engineering Technology SAFC Biosciences
Trevor Collingwood Ed Rebar Kevin Kayser
ZFNs Drive Site-Specific Targeted Insertion Yolanda Santiago Jeff Miller Jennifer Cresswell
into Native Endogenous Loci Salvatore Orlando Philip Gregory
Pei-Qi Liu Mike Holmes Sangamo Advanced
Homologous recombination (HR) is very inefficient in somatic cells (1 in 106). However, HR becomes >1000- Edmond Chang Genomics
-/- -/- -/- Sort GFP-positive cells then re-PCR
Targeting DHFR in a GS Cell Line Creates a GS DHFR fold more frequent during Homology-Directed Repair (HDR) of a DNA break. We make use of this by cleaving Greg Cost Sigma-Aldrich Research Shuyuan Yao
Double Gene Knockout the native locus with ZFNs at the desired site of integration, thus stimulating HDR. By providing the cell with Biotechnology Dmitry Guschin
a homologous donor plasmid that contains a desired transgene, we trick the cell into inserting the transgene Advanced Genomics Xiaoxia Cui Adam Waite
Treat GS-/- clone B3 with ZFNs targeting dihydrofolate reductase (DHFR) exon 1 Larger PCR product is enriched, Fyodor Urnov David Briner
at the site of cleavage. David Paschon
indicating that most GFP fluorescence
Dilution cloning gave 4/200 clones (2%) with both copies of DHFR disrupted Erica Moehle Gary Davis
The frequency of ZFN-mediated TI is usually >1%. Thus, antibiotic selection of clones is not required. is due to targeted insertion rather
than random events. Russ DeKelver Greg Davis
GS-/- DGFR-/- clones do not express GS or DHFR protein, and they require glutamine and HT for growth
04200-22104
* = attending conference 0028

04200 ECI poster.indd 1 3/6/2008 8:26:37 AM