2A used in Genome Editing Technologies.

Programmable nuclease platforms

Genome editing (GE) technologies based on custom designed nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) are opening up unprecedented possibilities for targeted changes within the genome of cells and organisms. The double-strand break made by these targetable nucleases stimulates the cell’s natural DNA-repair mechanisms, typically leading to one of two outcomes: non-homologous end joining (NHEJ) to introduce insertions/deletions, or if an appropriate target-homologous donor is supplied, homology directed repair (HDR). ZFNs and TALENs, although they share the same FokI-derived nuclease domain, differ in that they use distinctive DNA-binding arrays: ZFNs use zinc finger arrays and TALENs use TAL effector repeat arrays. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas RGNs rely on base pairing between a “guide” RNA and the DNA target for recognition and a Cas nuclease for DNA cleavage. While the nuclease triad has shown to facilitate GE, each approach has its pros and cons, which may dictate the choice for a given experiment.

What roles can 2A-like sequences play in genome editing technologies?

One of the major bottlenecks to the application of programmable nucleases is the lack of systems to select or enrich genome-modified cells. GE by using 2A peptide coupled co-expression of nuclease and fluorescent proteins combined with fluorescence-activated cell sorting (FACS) can aid selective enrichment of transfected cells (Ding et al., 2013; Xu et al., 2013; Joglekar et al., 2013; Mariano et al., 2014; Duda et al., 2014).

To improve the delivery of both ZFNs to the same cell in equal amounts using 2A-linked ZFN monomers, Joglekar and co-workers (2013) designed a single integrase-defective lentiviral vector encoding both the left and right ZFN monomers linked by the 2A peptide from Thosea asigna virus (T2A) and linked to mCherry via the 2A peptide from the porcine teschovirus-1 (P2A), to monitor gene transfer and expression by flow cytometry.

Because Fok1 functions as a dimer, TALENs, like ZFN’s, are designed in pairs that bind opposing DNA target sites. Perhaps the most obvious approach for this typoe of efficient genome editing is to improve the delivery of both TALEN monomers to the same cell in equal amounts using 2A-linked TALEN monomers. To this end, Mariano et al. (2014) compared the gene editing performances of co-transfected TALEN-L and TALEN-R monomer constructs versus a single construct encoding 2A-linked TALENs targeting two different loci: myostatin (MSTN) and AAVS1. TALENs targeting the MSTN gene expressed from one plasmid exhibited higher gene editing activity compared to co-transfection with the same TALENS in two separate plasmids (Xu et al., 2013; Mariano et al., 2014).

Selecting for Homology Directed Repair (HDR). Fluorescent reporter systems have also proven indispensable for evaluating the two major DNA repair pathways. The two-colour “Traffic Light Reporter” (TLR, comprising [eGFP-T2A-mCherry]) developed by Scharenberg and colleagues uses a flow cytometric assay to simultaneously detect both gene repair and mutagenic NHEJ at a single targeted site (Certo et al., 2011). HDR using a donor template carrying an intact eGFP results in green fluorescent cells. If mutagenic NHEJ repairs the break, a shift in the reading frame of the eGFP reporter can result in translation of the T2A-mCherry, leading to red fluorescent cells. In an independent study Kühn and co-workers adopted three different strategies (gene silencing, small-molecule inhibition or proteolytic degradation) to abolish NHEJ in a “traffic light”, leading to enhanced HDR for CRISPR-Cas9-induced gene targeting (Chu et al., 2015).

References citing the use of 2A;

Chu, V.T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K. & Kühn, R. (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnology 33(5), 543-548.

Duda, K., Lonowski, L.A., Kofoed-Nielsen, M., Ibarra, A., Delay, C.M., Kang, Q., Yang, Z., Pruett-Miller, S.M., Bennett, E.P., Wandall, H.H., Davis, G.D., Hansen, S.H. & Frödin, M. (2014). High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Research 42(10), e84 doi: 10.109/nar/gku251.

Mariano, A., Xu, L. & Han, R. (2014). Highly efficient genome editing via 2A-coupled co-expression of two TALEN monomers. BMC Research Notes 7:628.

Ding, Q., Lee, Y-K., Schaefer, E.A.K., Peters, D.T., Veres, A., Kim, K., Kuperwasser, N., Motola, D.L., Meissner, T.B., Hendriks, W.T., Trevisan, M., Gupta, R.M., Moisan, A., Banks, E., Friesen, M., Schinzel, R.T., Xia, F., Tang, A., Xia, Y., Figueroa, E., Wann, A., Ahfeldt, T., Daheron, L., Zhang, F., Rubin, L.L., Peng, L.F., Chung, R.T., Musunuru, K. & Cowan, C.A. (2013). A TALEN genome editing system to generate human stem cell-based disease models. Cell Stem Cell 12(2), 238-251.

Joglekar, A.V., Hollis, R.P., Kuftinec, G., Senadheera, S., Chan, R. & Kohn, D.B. (2013). Integrase-defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus. Molecular Therapy 21(9), 1705-1717.

Xu, L., Zhao, P., Mariano, A. & Han, R. (2013). Targeted myostatin gene editing in multiple mammalian species directed by a single pair of TALE nucleases. Molecular Therapy Nucleic Acids 2(7): e112.

Certo, M.T., Ryu, B.Y., Annis, J.E., Garibov, M., Jarour, J.V., Rawlings, D.J. & Scharenberg, A.M. (2011). Tracking genome engineering outcome at individual DNA breakpoints. Nature Methods 8(8), 671-676.

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