SplitAx:A novel method to assess the function of engineered nucleases

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization

Schematic diagram illustrating the different mechanisms of how the SplitAX assay functions.

<p>The vector consisting of the pCAG promoter, the GFP cDNA (N-terminus 1-474bp), a genome editing binding site containing a stop codon which is out of frame with the GFP cDNA C-terminus (475-end). In the absence of exposure to a specific genome editing tool, the full length GFP protein is not expressed. Exposure of the GFP-SplitAx to a genome editing tool creates a double strand break. Repair by non-homologous end joining (NHEJ) mutates the binding site restoring the open reading frame (ORF) of GFP resulting in fluorescence. The second mechanism involves the repair of the double strand break by NHEJ resulting in an N-terminal ORF in frame with the C-terminal GFP. The C-terminal GFP can complement with the N-terminal GFP expressed from a different vector leading to restored fluorescent activity.</p

Functional validation of the ZsGreen1-AAVS1 SplitAx reporter assay with AAVS1 zinc fingers.

<p>Schematic of the ZsGreen1 cDNA with the N-terminus and C-terminus separated by the AAVS1 binding site. The DNA sequence of the AAVS1 binding site is shown and the location of zinc finger left (ZF L), Zinc finger right (ZF R) (a). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-AAVS1 SplitAx only (b), ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Left (Zn L) (c), ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Right (Zn R) (d), and ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Left/Zinc Finger Right (Zn L, Zn R) (e). Graphical representation of flow cytometry data for the ZsGreen1-AAVS1 SplitAx with the AAVS1 Zinc Fingers (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (f).</p

Functional validation of the ZsGreen1-Cfms-SplitAx reporter assay with Cfms gRNAs and hCAS9 or D10A nickase.

<p>Schematic diagram of the 5’ and 3’end of Zs Green1 separated by the <i>Cfms</i> binding site. The DNA sequence of the Cfms binding site is shown and the location of the gRNA_Cfms-8a, 8b and 9b are underlined (a). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx with hCAS9 (b), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8a (c), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8b (d), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-9b (e). Quantification of flow cytometry data for the ZsGreen1-Cfms- SplitAx and hCAS9 with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (f). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx only (g), ZsGreen1-Cfms-SplitAx with D10A nickase (h), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and8b (i), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and 8b (j). Graphical representation of flow cytometry data for the ZsGreen1-Cfms- SplitAx and D10A nickase with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (k).</p

Functional validation of the GFP-AAVS1 SplitAx reporter assay with zinc fingers and CRISPR/CAS9 system.

<p>Schematic of the GFP cDNA with the N-terminus and C-terminus separated by the <i>AAVS1</i> binding site. The DNA sequence of the <i>AAVS1</i> binding site is shown and the location of zinc finger left (ZF L), Zinc finger right (ZF R), <i>AAVS1</i> guide RNAs T1 and T2 underlined (a). The translated DNA sequence of the <i>AAVS1</i> binding site with stop codons (-) (b). The translated DNA after genome editing. In this case a 1 bp deletion removes the stop codons and allows in frame of translation of the C-terminal GFP resulting in fluorescence (c). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (d), GFP-AAVS1 SplitAx with single AAVS1 Zinc Finger Left (Zn L) (e), GFP-AAVS1 SplitAx with AAVS1 single Zinc Finger Right (Zn R) (f), and GFP-AAVS1 SplitAx with both AAVS1 Zinc Finger Lefand/Zinc Finger Right (Zn L, Zn R) (g). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the AAVS1 Zinc Fingers (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (h). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (i) GFP-AAVS1 SplitAx and hCAS9 (j), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T1 (k), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T2 (l). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the CRIPSR gRNA_AAVS1- T1 or T2 and hCAS9 (+), cells not transfected with a plasmid (-). Data shown as +STDev (n = 3) (m).</p