FP-ZFNs or Fluorescent Protein-Linked Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) have enabled highly efficient gene targeting in a wide variety of cell types and organisms. While CompoZr® methods for engineering highly active ZFNs have become robust and reproducible, challenges relating to ZFN delivery and expression continue to limit isolation of correctly edited clones for certain cell types.

Previous work has addressed ZFN delivery and expression problems by implementing drug selection strategies with co-delivered donor DNA plasmids. Other enrichment approaches have relied on ZFN modification of co-delivered fluorescent protein reporters that are activated upon ZFN cutting. These methods require construction of a reporter plasmid harboring the ZFN cut site, and are limited to dsDNA formats which (1) can adversely affect certain cell types that are sensitive to dsDNA, and (2) create a high risk of random reporter gene integration into the genome during the selection process.

Sigma presents a simple method that is highly effective at enriching correctly edited cell populations by co-expressing ZFNs and fluorescent proteins from the same in vitro transcribed mRNA. When combined with ssDNA donors, a “dsDNA-free” mode of genome editing becomes possible.

Monitoring cellular delivery of ZFN mRNA and plasmids

Alternate ZFN vector formats provide a fluorescent protein (FP) between the promoter and start of the ZFN coding region. Cells which have been transfected with FP-linked ZFN vectors can be subsequently inspected by microscopy or FACS to monitor transfection efficiency and ZFN expression levels. 

Enriching cell populations for ZFN-modified cells using fluorescent reporters

If you are using ZFN expression vectors which encode a fluorescent protein (FP), fluorescence-activated cell sorting (FACS) can be used to isolate cell populations with significantly increased frequencies of ZFN-induced modifications.  This FACS-enrichment approach is particularly useful in scenarios where delivery efficiencies and/or ZFN expression levels are low.  Within CompoZr FP-ZFN expression vectors, the FP and ZFN protein coding regions are linked by a small sequence encoding a 2A-peptide. The 2A peptide is a ‘‘self-cleaving’’ peptide which allows production of two individual proteins from one transcript and utilizes “ribosomal skipping” rather than proteolytic cleavage mechanism to generate two individual proteins. The average length of 2A peptides is approximately 18–22 amino acids (Ryan et al., 1991; Donnelly et al., 2001). In CompoZr FP-ZFN expression constructs, TagGFP2 or TagRFP (hereafter referred to as GFP or RFP) is linked to the N-terminus of the left ZFN (ZFN1) or right ZFN (ZFN2) by the 2A peptide.  

Map of FP-ZFN Vectors

Tips for FACS enrichment of cells expressing FP-ZFNs

Note 1: ZFNs in plasmid or mRNA form can be used for delivery to cells, although it is recommended that ZFN mRNA be used since some cell types are sensitive to dsDNA delivery. 

Note 2: If mRNA is used, cells should be incubated at 30˚C for 2-3 days after nucleofection or transfection. Cold-shock will maximize FP-ZFN expression from the transcript and has been found to be a critical if mRNA is to be used for delivery. If plasmid DNA is used, cells can be incubated at 37˚C or 30˚C. Following delivery and prior to FACS sorting or single cell cloning, cells should be checked by microscopy to observe fluorescence. 

Note 3: Cells can be harvested for FACS according to commonly used protocols. We suggest that cells be sorted into low, medium, and high FP expression levels in regions of equivalent GFP and RFP expression. Cell populations with the highest GFP/RFP expression have been shown to enrich genome edits with the extent of the “high” fraction ranging from 1 to 30% of the total cell population. The optimal size of the “high” and “medium” populations may vary, depending on particular ZFNs, genomic loci, cell types, delivery methods, and other experimental variables. A good start is to divide the cell population into three low, medium, and high fractions each of which comprises 1 to 20% of the total cell population.

Properties of fluorescent proteins in CompoZr ZFN vectors

Sigma FP-linked ZFN expression vectors use Evrogen’s TagGFP2 and TagRFP fluorescent proteins. TagGFP2 is the improved variant of TagGFP, a mutant of the Aequorea macrodactyla GFP-like protein (Xia et al., 2002, Subach et al., 2008). TagGFP2 possesses bright green fluorescence with excitation/emission maxima at 483 and 506 nm, respectively. TagRFP is a monomeric red (orange) fluorescent protein generated from the wild-type RFP from sea anemone Entacmaea quadricolor (Merzlyak et al., 2007). It possesses bright fluorescence with excitation/emission maxima at 555 and 584 nm, respectively (Shaner et al., 2004).

FP-ZFN Data

We present a general and simple method in which a fluorescent protein (FP) is linked to a ZFN by the 2A peptide, allowing for coupled (1:1) co-expression of fluorescent protein and ZFNs in the same cells followed by isolation of nucleofected cells by fluorescence-activated cell sorting (FACS). Our approach enables easy isolation of cell populations that express similar levels of ZFN in hard-to-transfect cells and enrichment of ZFN activity via FACS, thus greatly improving ZFN-based genome editing or cell cloning.

Figure 1. Schematic of fluorescent protein-linked ZFNs. GFP is linked to the N-terminus of left ZFN (ZFNL) and RFP to the N-terminus of right ZFN (ZFNR) by a 2A peptide sequence, respectively.

 

Figure 2. ZFN transfection efficiency can be examined via fluorescence microscopy and FP-ZFN vectors preserve ZFN activity relative to non-FP ZFN vectors. Human K562 and Jurkat cells were nucleofected with ZFNs expressed from an eHiFi vector and FP-linked vector targeting AAVS1 site in forms of DNA (plasmid) and mRNA as indicated. Cells were examined and photographed under fluorescence microscope (K562 cells, X40 magnitude) two days after nucleofection (A), and harvested for CEL-I assay to evaluate ZFN cutting activity (B).

 

Figure 3. FACS enables the enrichment of ZFN activity and rescue of undetectable ZFN activity. Jurkat cells were nucleofected with ZFN mRNAs targeting human AAVS1 locus or RSK2 kinase gene and subjected to FACS two days after nuclofection. Three groups of cells were pooled based on the signal intensity of fluorescent proteins (low, medium and high), which were subjected to the CEL-I assay to evaluate the cutting activity. (A & C) FACS profile of Jurkat cells nuclefected with ZFN mRNAs targeting the human AAVS1 and RSK2, respectively; (B & D) CEL-I assay of pooled FACS isolated population for ZFN mRNA targeting AAVS1 and RSK2 loci, respectively.

 

Figure 4. FP-ZFNs can be combined into single vector format for FACS enrichment of ZFN activity. Jurkat cells were nucleofected with ZFN mRNA or GFP control plasmid as indicated. Cells were harvested and subjected to CEL-I assays two days after nuclefoection. (A) Schematic of GFP-tagged ZFNs in a single vector. GFP is linked to the left ZFN (ZFNL) and right ZFN (ZFNR) by a 2A peptide sequence. Restriction endonuclease sites are indicated; (B) FACS profile after nucleofection of Jurkat cells with mRNA for GFP-2A-ZFNL-2A-ZFNR targeting the human RSK2 locus. Cells from populations displaying varying fluorescence intensities (low, medium, high) were FACS isolated and pooled for the CEL-I mismatch detection assay; (C) CEL-I assay of the pooled FACS isolated cell population.

 

Figure 5. FACS improves the efficiency of oligo-based target gene integration. K562 cells were nucleofected with FP-ZFN mRNA targeting human HBB gene with or without an oligo that contains an AcuI restriction endonuclease site (ssODN). Cells were FACS sorted and harvested two days after nucleofection for CEL-I assays of pooled cell populations and RFLP analysis of target integration efficiency. (A) The schematic of the 80-mer single-stranded donor DNA used to incorporate an AcuI site in the HBB genomic locus; (B) FACS profile after nucleofection of K562 cells with ZFN mRNA and oligo. (C) CEL-I assay of pooled FACS isolated cell population. (D) AcuI RFLP analysis of PCR product of pooled FACS isolated cell population and the efficiency of AcuI cleavage quantified by densitometry.

 

Figure 6. Nick-oligo genome editing occurs at low efficiency, but can be enriched to detectable levels in FP-ZFNickase format. K562 cells were nucleofected with FP-tagged ZFNickase mRNA targeting human AAVS1 locus with or without an oligo that contains a HindIII restriction endonuclease site (ssODN). Cells were FACS sorted and harvested two days after nucleofection for CEL-I assay of pooled FACS isolated population and HindIII RFLP analysis of target integration efficiency. (A) The schematic of the 95-mer single-stranded donor DNA used to incorporate a HindIII site in the AAVS1 locus; (B) FACS profile after nucleofection of K562 cells with ZFNickase mRNA and oligo; (C) CEL-I assay of pooled FACS isolated cell population; (D) HindIII RFLP analysis of PCR product of pooled FACS isolated cell population and the efficiency of HindIII cleavage quantified by densitometry.

FP-ZFNs are Now Available as a Custom ZFN Option

You can add the FP-ZFN vector option to any custom ZFN or customize any Knockout ZFN by contacting your local Sigma-Aldrich sales representative or fill out this form  and check yes to the question “Add Fluorescent Protein Linked ZFN vector?”

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