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Biowire Fall 2010 — A Look into the Future of Gene Editing

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ZFN Donor Design — Codon and Single Base Genome Editing Using Zinc Finger Nucleases

A guide for the design of targeting donors for creating point mutations, codon changes, SNP corrections, and other small site-directed genomic modifications

ZFN Design and Positioning Relative to the Desired Mutation Site
Length of Homology Arms and Plasmid Backbone
Detecting desired mutations in pooled and clonal cell populations
Preventing unwanted NHEJ mutations when creating precise point mutations
Detecting mutations at the pooled and clonal cell level

ZFN Design and Positioning Relative to the Desired Mutation Site
Homologous recombination (HR) is a process by which double-stranded breaks (DSBs) are repaired in cells. In the context of normal cellular function, HR is a "copy and paste" mechanism by which a DSB is repaired using the information contained in a homologous sister chromatid. However, a user-defined mutation can be introduced by transfecting a donor plasmid that contains both the userspecified mutation and sequence homologous to the regions flanking the desired site of modification. In mammalian cells, the spontaneous rate of HR between a userdefined donor plasmid and a homologous locus is very low (10–6 to 10–5) and is often outside the range for practical experimentation, even when using antibiotic selection. However, several groups have shown that the creation of a DSB at or near the site of desired mutation can increase the rate of HR by several orders of magnitude (Rouet, et al., 1994). Until recently, the creation of a DSB at a desired locus was not feasible. Zinc finger nucleases (ZFNs) are engineered endonucleases that can now be rapidly designed to create DSBs in the vicinity of a desired mutation site with routine success (Urnov, et al., 2005). Following successful design and manufacturing of a site-specific ZFN, the next key step to create targeted mutations via HR is to design and construct a targeting donor plasmid.

An overall workflow for donor plasmid design is shown in Figure 1. If the goal is to create a point mutation, the ZFN should be designed to cut within 200 bp of the desired mutation site. Existing literature (and unpublished data) shows that the penalty for moving the point mutation 100 bp away from the cut site is an approximate 4X drop in mutation frequency (Elliot, et al., 1998). These authors documented a point mutation 511 bp from a nuclease cut site, however, it occurred at a frequency that would likely require screening of >1,000 clones to isolate a mutant in the absence of selection. So, whenever possible, it is advisable to use a ZFN that is located <200 bp away from the cut site to maintain high efficiency and thus minimize clone screening. Using selection methods, it is certainly reasonable to expect that mutations can be incorporated >500 bp from the cut site, but these applications require position-restricted integration of large selectable gene cassettes that complicate ZFN design and risk affecting gene expression and regulation.

Biowire Fall 2010 ZFN Donor Design Figure 1

Figure 1. Workflow for designing plasmids to create small mutations near ZFN cut sites.

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Length of Homology Arms and Plasmid Backbone
When designing donor plasmids for making small mutations, it is recommended to use ~400-800 bp of homology in each arm centered close to the ZFN cut site (Figure 2). Donor plasmids with 800 bp of homology in each arm have been shown to work robustly in different applications requiring point mutations. Donor plasmids with shorter arm lengths (50-100 bp) have been shown to work (Orlando, et al., 2010), but are generally less effective than 400-800 bp across a broad range of cell types. It is reasonable to assume that selection methods might enable use of 50 bp homology arms in a broader range of cell types, but this has yet to be explored.

For plasmid backbones, it is recommended to use small pUC-based plasmids (<2,600 bp) to limit the mass of DNA that is transfected to cells. To date, multiple variations of pUC based backbones have been used to successfully execute ZFN-based gene targeting experiments. Total gene synthesis companies (that can be used for donor synthesis) generally prefer plasmid backbones which minimize the cost of their cloning and sequencing operations. It is perfectly fine to use whatever standard cloning vector vendors offer as long as the plasmid is small, lacks common restriction sites, and lacks significant sequence homology to the target region of interest.

Biowire Fall 2010 ZFN Donor Design Figure 2

Figure 2. Example donor plasmid for creating a codon change near a ZFN cut site. Three key mutations need to be considered: (A) a silent mutation that allows detection and quantification via PCR and restriction enzyme cleavage (RFLP), (B) the desired mutation for the biological experiment, and (C) mutations (ZBMs) within the ZFN binding site that disrupt intracellular ZFN cleavage of the plasmid or cleavage of the chromosome post-integration of the donor plasmid. Note: the desired mutation (B) should be flanked by the detection mutation (A) and the ZFN cut site. In rare cases, the desired mutation will create a new unique restriction site.

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Detecting desired mutations in pooled and clonal cell populations
Prior to investing the effort to derive and screen a clonal cell population, it is useful to estimate the mutation frequency at the pooled cell level immediately following transfection of the ZFN and donor plasmid. Addition of a silent restriction site somewhere in the donor will facilitate easy detection of mutant clones (e.g., an RFLP site). The RFLP site should be as close as possible to the desired mutation and always positioned so that the desired mutation is flanked by the RFLP site and the ZFN cut site (see Figure 2A). Homologous recombination inserts new sequence directionally in relation to the ZFN cut site, and this method of positioning the RFLP site will help ensure that when analyzing clones the desired mutation is present in all RFLP positive clones. In rare cases, the desired mutation may actually create a unique restriction site. An excellent resource for finding silent mutations quickly is WatCut: (http://watcut.uwaterloo.ca/watcut/watcut/template.php).

When creating point mutations to create RFLP sites and amino acid changes, it is recommended to check the codon usage in the gene of interest and to use the most frequently used codons in that gene where feasible. In rare cases, even silent mutations can have drastic effects on functional gene expression (Kimchi-Sarfaty, et al., 2007). A good site for determining codon usage frequency is: http://www.ebioinfogen.com/biotools/codon-usage.htm. After finding a good RFLP site, check that it is not located at another site within the donor, since this will complicate downstream analysis.

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Preventing unwanted NHEJ mutations when creating precise point mutations
If the ZFN cut site is present within an open reading frame (ORF) and the goal is to make a nearby codon change or SNP correction, it is possible that the ZFN will also cause undesirable secondary mutations at the cut site via misrepair arising from non-homologous end joining (NHEJ). In our work with donor plasmids, some configurations resulted in 30-40% of the correctly modified clones also having secondary mutations at the ZFN cut site, while other applications had >90% secondary mutations. The level of secondary mutations appears to be higher when the desired mutation is located further away from the cut site. As a general practice, we recommend incorporating silent mutations in the ZFN binding site to prevent ZFN cleavage of the donor plasmid and/or recleavage of the target site post-integration of the plasmid. At least two ZFN-blocking mutations (ZBMs) should be incorporated into the ZFN binding site - separated by at least 3 bp if they are within the same ZFN arm, or one placed in each arm (Figure 2C). The 3 bp spacing ensures each ZBM lies in the binding sequence of a different zinc finger, thereby maximizing the chances for disruption of binding. These mutations should be selected in a way that minimizes changes in codon usage frequency. This can be done by placing the ZBMs at amino acids with highly degenerate codons, such as Ala, Val, Thr, Pro, Ser, Leu, Gly, and Arg. This will give more options for preserving codon usage frequency since they all have approximately four different codons to select from.

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Detecting mutations at the pooled and clonal cell level
Following transfection of the ZFN and donor construct, the next step is to assess the rate of mutation in the pooled cell population prior to diluting (or FACS sorting) the cells for single cell cloning. Two different methods maybe used to detect mutations: (1) RFLP, using a silent restriction site (Figure 3), and/or (2) mutation-specific junction PCR (Figures 4A and 4B). Because both assays are based on PCR, it is essential to always include a "donor only" transfected sample to control for PCR artifacts, especially at the pooled level when copy number of the donor plasmid will be high. For analyzing a pool of cells, the RFLP approach is the most quantitative method and best predicts success in follow-up single cell cloning efforts. For RFLP detection, two PCR primers should be designed that prime outside the region of homology present on the donor plasmid (out-out PCR; Figure 3). Genomic DNA is isolated 2-3 days posttransfection and used as a PCR template. The PCR product is then digested with the RFLP restriction enzyme (in this case BamHI) and resolved by electrophoresis on an agarose or polyacrylamide gel. The rate of targeted integration can be estimated by performing densitometry on the gel and comparing the intensity of the fragments released by restriction digestion against the intensity of the parental band. If the mass of the parental PCR band is in excess of 700 ng, then the signal from the band is likely saturated and out of the linear range required for accurate quantitation. In our experience, RFLP signals that are visually observable by EtBr staining, regardless of parental band intensity, have high chances of success. Figure 3 shows a good example of overloading of the parental PCR product, with observable RFLP fragments. Despite overloading, this experiment yielded correctly targeted biallelic K562 clones at a rate of 6 of 112 clones screened.

Biowire Fall 2010 ZFN Donor Design Figure 3

Figure 3. Detection of modified alleles by RFLP (e.g., out-out PCR) at 2 days post-transfection of ZFNs and donor plasmid.

When screening clones, either the RFLP or junction PCR approach may be used. Figures 3-5 outline an experiment in which RFLP was performed on the transfected cells (Figure 3), then "out-in" junction PCR was used to screen clones to identify those with the ZBM mutation introduced at the cleavage site (Figure 4A and 4B). A subset of these ZBM-positive clones will also contain the desired mutation. Finally, RFLP was used (Figure 5) to assess whether selected clones were monoallelic or biallelic for the BamHI conversion. When screening clones, the RFLP assay becomes more stochastic in that a small defined number of modified alleles will contribute to the RFLP signal (2 to 6 alleles, depending on ploidy). The typical experimental goal is to find a clone that completely lacks wildtype sequence, resulting in complete digestion of the parental PCR product (Figure 5, e.g., clone B7), though heterozygous mutations maybe biologically relevant depending on circumstances. Following confirmation of biallelic (or complete) conversion by RFLP, the fidelity of the mutation should be confirmed via DNA sequencing of all alleles. DNA sequencing of clones showing biallelic BamHI conversions revealed that all alleles containing a BamHI conversion also contained the desired codon mutation. This result provides further justification for always flanking the desired mutation by a silent RFLP and the ZFN cut site as illustrated in Figure 2.

Biowire Fall 2010 ZFN Donor Design Figures 4 and 5

Figure 4-B. Detection of modified alleles by junction PCR using a mutation specific primer (e.g., out-in PCR).   Figure 5. Detection of modified alleles by RFLP (e.g., out-out PCR) of individual clones at 3-weeks post-transfection of ZFNs and donor plasmid.

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References

Gene conversion tracts from double-strand break repair in mammalian cells. Elliott, B., et al., Molecular & Cellular Biology, 18 (1), 93-101 (1998).

A "silent" polymorphism in the MDR1 gene changes substrate specificity. Kimchi-Sarfaty, C., et al., Science, 318 (5811), 525-8 (2007).

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors within limited chromosomal homology. Orlando, S.J., et al., Nucleic Acids Research (2010).

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Rouet, P., et al., Molecular & Cellular Biology, 14 (12), 8096-106 (1994).

Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Urnov, F.D., et al., Nature, 435 (7042), 646-51 (2005).

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