What is a Zinc Finger Nuclease (ZFN)?
Why are these sometimes referred to as ZFN pairs?
What specificity should be expected from a ZFN?
Will a ZFN knockout a gene in my cell line?
Will a ZFN insert a gene into my cell line?
What is the best way to deliver the ZFN and the repair template into the cells?
What are the advantages to delivering ZFNs to cells in mRNA form?
What steps are required to produce a validated ZFN?
How is the binding specificity of the ZFN tested?
How has the cleavage ability of my ZFN been tested?
In which cell lines/organisms do the ZFNs work?
I have a cell line I created by using a ZFN to knock-out a gene. I would like to knockout a second gene in this cell line. Is this possible?
Can every gene be targeted by a ZFN?
Should I expect off-target effects from my ZFN?
My cell line is very hard to transfect. Can I still use ZFNs?
Do the ZFNs remain in the cells after causing desired mutations?
Do I need to use a selection marker for Targeted Gene Integration?
A ZFN is a hybrid molecule that couples the DNA binding domain of a zinc-finger protein with the DNA-cleaving nuclease domain of the restriction endonuclease FokI. The DNA binding motif specified by the zinc fingers directs the ZFN to a specific (targeted) locus in the genome.
A pair of ZFNs is required to cleave double-stranded DNA. This is a requirement of the FokI nuclease. FokI must dimerize to achieve a double strand break in the DNA. You will be provided with a pair of ZFNs for your target site that has been validated to cut at the endogenous chromosomal locus in a proxy cell line.
Due to the dimerization requirement of the FokI endonuclease, a pair of ZFNs is required to cause a double strand break. This strategy requires two different ZFNs to bind at the target site. Each ZFN recognizes a different 12-18 base pair target sequence, and these target sequences must be separated by 4-7 base pairs to allow formation of the catalytically active FokI dimer. These positional constraints drive a very high degree of specifi city.
Yes. ZFNs for gene knockout typically target within the ORF of coding exons. The ZFN that is delivered to you for gene knockout will cause a targeted double strand breakage (DSB) in the genome of your cells. After the DNA is cut by the ZFN, the cell repairs the breakage by the natural process of non-homologous end joining (NHEJ). This is an imperfect repair process that usually results in the loss or gain of genetic information (typically tens of base pairs) at the site of the DSB. Due to the mutations caused by ZFN-mediated NHEJ, 1–20% of resulting clones are expected to have both copies of the target gene knocked out.
Yes. To achieve targeted gene integration you must co-transfect your ZFN with a repair template, consisting of the gene to be inserted flanked by genomic sequence engineered to match (i.e. be homologous to) the sequence surrounding the intended ZFN cut site. After your ZFN cuts at the site specified by its sequence, the natural mechanism of homology dependent repair (HDR) takes over. The cell will recognize the homologous sequence in the repair template and insert the exogenous gene at the site of ZFN cleavage.
Repair templates are typically co-delivered into the cell as circular plasmid DNA. The ZFN itself can be delivered as a plasmid, or as an mRNA transcript. We have found both plasmid and mRNA to be effective depending on cell type and, therefore, provide customers with ZFNs in both plasmid and mRNA form to test in their own cell lines. Regardless of form, we have found that the use of nucleofection or electroporation generally produce the highest cleavage efficiencies. Due to this increase in efficiency, we recommend using an electroporation or nucleofection delivery method if possible. Lipid-based transfection also works in many cases, but may result in lower efficiencies.
There are many advantages to using a ZFN mRNA transcript. We have found the following benefits in a number of tested cell types:
We go through the following steps to produce a validated ZFN for our customers:
The binding specificity of the zinc finger modules in the Sangamo archive have previously been characterized by a number of in-vitro methods (ELISA among others).
To test the cleavage ability of the ZFNs we use a mismatch-specific nuclease assay. In this assay, PCR primers are used to amplify across the ZFN cleavage site in genomic DNA extracted from ZFN-treated cells. The resulting PCR products, which are now a mixture of wild type and mutated amplicons, are then denatured and re-annealed. When wild type and mutant alleles of the PCR products re-anneal with each other a mismatch occurs. The fragments are then exposed to a mismatch-specific nuclease and the digested reaction is run on a gel to look for smaller migrating cleavage products that indicate cleaved mismatches. The smaller migrating cleavage products reflect the frequency of mutated alleles and are a measure of in vivo ZFN efficacy. Quantitation of the released fragments allows a calculation of the percentage of mutated genes in the population.
We have a standard offering of ZFNs for Human, Mouse, and Rat. We expect the ZFN technology to work in most organisms. For instance, ZFNs have recently been shown to produce site-specific gene knockouts in Zebrafish (Doyon et al. Nature Biotechnology May 25, 2008). The ZFNs for this particular application were tested in a yeast proxy system that accurately reflects ZFN activity in many other cell types.
Yes, this is definitely a possibility. It has been shown that at least 3 different genes could be successively knocked out of the genome in the same cell line.
Yes. Using our current library of 2-finger zinc finger modules, we believe we can produce a working ZFN for about every 50 bp on average, making genome wide coverage feasible. We plan to continue updating our library of zinc finger modules to increase the genomic coverage.
The ZFN reagents that we provide are unlikely to cause significant offtarget effects. Our proprietary algorithm for candidate ZFN design only targets unique genome sites. This, as well as the use of specially engineered obligate heterodimer FokI cleavage domains (Miller et al. Nature Biotechnology July 25, 2007), helps guard against off-target effects for the ZFN you receive.
Efficiency of delivery is very important. We have had great success with viral delivery of ZFNs, including AdV, AAV and lentivirus. If you have a viral delivery system available, then we can give guidance on how to apply it to the use of ZFNs.
No. The ZFNs are only expressed transiently but the genetic alteration is permanent. The transient nature of ZFNs also reduces the possibility of off-target effects.
In many cases, the efficiency of Targeted Gene Integration is >1% — even without selection. So selection markers are often not necessary. However, in certain circumstances, selection might be useful to have, e.g., when working with poorly transfectable cells.