Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair

Abstract

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.

Keywords: CRISPR/Cas9; homology-directed repair; precise gene editing; promoterless EGFP reporter; random integration.

Figure 2

Porcine cells transfected with different forms of reporter. (A) Structure of the reporter digested with AhdI, EcoRI, EcoRI+HindIII, respectively. (B) PFF and (C) PK15 cells were transfected with different forms of reporter donors. BC: Blank control, BC group is no treatment cells. EGFP control: cells transfected with EGFP-N2 plasmid were as positive control. Reporter plasmid: circular reporter plasmid only. Reporter ^EcoRI: reporter was digested with EcoRI. Reporter ^{EcoRI+HindIII}: reporter was digested with EcoRI and HindIII. Scale bar: 50 μm.

Figure 5

Validation the EGFP expression in 293T cells transfected with different forms of reporter. (A) Structure of the reporter digested with NsiI, BbsI, respectively. (B) Fluorescence microscopy and flowcytometry analysis of 293T cells transfected with different forms of reporter at 48 h post-transfection. Reporter plasmid, reporter digested with BbsI (Reporter ^BbsI) or reporter digested with NsiI (Reporter ^NsiI) were transfected into 293T cells, respectively. Scale bar: 50 μm.

Figure 1

Overview of HDR reporter targeting the GAPDH locus. (A) Schematic of GAPDH targeted HR reporter strategy. The CRISPR/Cas9 targeting the stop codon (TAA) of GAPDH exon 12. Reporter vector was designed containing an 800-bp left homologous arm cover the exon 8, 9, 10, 11 and 12 upstream from the stop codon, and a 500-bp right homologous arm downstream of the stop codon. (B) Validation intracellular indels efficiency and indels pattern of sgRNA3 targeting exon 12 of porcine GAPDH by TIDE analysis. Cells were transfected with CRISPR/Cas9 system and harvested after 48 h. (C) Fluorescence microscope of PFF and PK15 cells transfected with CRISPR/Cas9 system and reporter digested with AhdI (Reporter ^AhdI) or Reporter ^AhdI only 48 h post transfection. Scale bar: 50 μm.

Figure 3

Validation the reporter in non-porcine cells. Fluorescence microscopy and flowcytometry analysis of cells transfected with different forms of reporter at 48 h post-transfection, which involves in (A) CHO-K1, (B) HepG2 and (C) HepaRG. Scale bar: 50 μm.

Figure 4

Validation the reporter plasmid’s specificity in 293T. (A) 293T cells was transfected with different reporter as described in Figure 2. Scale bar: 50 μm. (B) Fluorescence observation of 293T transfected with reporter digested with EcoRI and HindIII every other two days. p.t. stands for post transfection. Scale bar: 50 μm. (C) Schematic of PCR primer designed for precise KI and transgene identification. The precise KI primer was used to validate the hypothetical reporter precise KI in human GAPDH. Transgene primer was designed within the cargo genes, that were EGFP and NeoR. The amplified products of forward primers from exon 8 and reverse primers from exon 9 of human GAPDH locus were taken as control.