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Delivery of FP-ZFNs (Fluorescent Protein-Zinc Finger Nucleases) using integration-deficient lentivirus (IDLV)

Background

Efficient delivery of ZFNs to the cell type of interest is required for modifying the cell’s genome. Lentiviral vectors offer an efficient delivery system, able to infect dividing and non-dividing cells, neuronal cells, terminally differentiated cells and stem cells. The new Sigma CompoZr FP-ZFN-IDLV products provide researchers with a lentiviral delivery format to increase the efficiency of genome modifications in various cell types, including hard-to-transfect cells. To preserve the transient mode of ZFN expression typically implemented in genome editing workflows, we use a system that enables integration-deficient lentiviral (IDLV) packaging to minimize integration of ZFN transgenes into the host genome.  Previous reports show that delivery of ZFNs via IDLV supports increased rates of gene editing (http://www.ncbi.nlm.nih.gov/pubmed/17965707, http://www.ncbi.nlm.nih.gov/pubmed/23857176) in various cell types. As an additional alternative approach to overcoming low delivery and expression levels in particular cell types, Sigma has created vectors in which ZFN expression is linked to fluorescent reporters.

ZFN-mediated Targeted Genome Editing

*Mutation due to addition or deletion of bases.

Figure 1. ZFN-mediated Targeted Genome Editing – A) ZFNs bind a DNA sequence of 30-36bp, inducing a specific double strand break (DSB). B) The DSB is repaired by NHEJ (non-homologous end joining), which is an error-prone process of repair.  

Lentiviral titers are known to be negatively impacted by large genetic payloads, and subcloning of repetitive sequences into lentiviral vectors has often resulted in vector recombination and instability. Due to their compact protein structure and low content of repetitive sequence, ZFNs have been successfully expressed and implemented using lentiviral vectors.

Integration-deficient lentivirus (IDLV) has been required to avoid permanent integration of ZFN transgenes into the host genome.  In general, relative to integration competent lentivirus, IDLV-formatted gene expression has lower transgene expression levels (http://www.ncbi.nlm.nih.gov/pubmed/19491821). To address these expression limitations, we have constructed vectors that link ZFN expression directly to fluorescent protein reporters.  This reporter-linked expression format enables enrichment of cell populations that have undergone both efficient transduction and subsequent high level expression of ZFN transgenes and stand to substantially increase genome editing frequencies. 

FP-ZFN Data

ZFN activity can be increased to frequencies required for subsequent single cell cloning. Using FP-formatted ZFN expression, ZFN activity at the RSK2 locus was successfully restored to detectable levels (>1% by CEL-I assay). 

Schematic of fluorescent protein-linked ZFNs

Figure 2. Schematic of fluorescent protein-linked ZFNs (A). GFP is linked to the N-terminus of left ZFN (ZF-FokI-ELD) and RFP to the N-terminus of right ZFN (ZF-FokI-KKR) by a 2A peptide sequence. (B) ZFN expression cassette activity can be rapidly monitored post-nucleofection via microscopy (K562 cells).

 

FACS enables sufficient enrichment to rescue undetectable ZFN activity

Figure 3. FACS enables sufficient enrichment to rescue undetectable ZFN activity. (A) FACS profile of Jurkat cells nucleofected with ZFN mRNAs targeting the human RSK2.  (B) CEL-I assay of pooled FACS isolated population for ZFN mRNA targeting the RSK2 locus. The CEL-I assay is a nucleotide mismatch assay used to detect the presence of mutant clones and quantify the effectiveness of ZFNs. The detected cutting efficiency of ZFNs was 9.1% in the high sort cells.

 

FP-ZFN-IDLV Data

The FP-ZFN approach is compatible with lentiviral transduction experiments, allowing more rapid and accurate assessment of the performance of lentiviral ZFN expression control elements. This reporter-linked expression format enables enrichment of cell populations and can substantially increase genome editing frequencies in clonal cell populations.

FP-IDLV-ZFN vector schematic

Figure 4. FP-IDLV-ZFN vector schematic.


FP-ZFNs-IDLV targeting H2AFX, U2OS cells were sorted (upper gate)

Figure 5. FP-ZFNs-IDLV targeting H2AFX, U2OS cells were sorted (upper gate). (A) ZFN expression cassette activity monitored post-nucleofection via microscopy. (B) U2Os cells were pooled sorted (upper gate), and ZFN cutting efficiency determined by the CEL-I assay (C). The CEL-I uncut wild-type band is 398bp, and the ZFN modified bands are 234 bp and 164 pb.

 

MSC Cells

Figure 6. MSC cells are hard to transfect cells that when nucleofected with ZFNs in plasmid format they undergo complete apoptosis within 24 hours. (A) MSC cells show good viability and visible modification when treated with ZFNs in IDLV format. (B) MSC cells were pooled sorted (upper gate), and ZFN cutting efficiency determined by the CEL-I assay (C). The detected cutting efficiency of ZFNs was 9 times greater in the high sort cells compared to pooled cells. The CEL-I uncut wild-type band is 398bp, and the ZFN modified bands are 234 bp and 164 pb.