Background

Genome editing tools are very important for the investigations of biological mechanisms and prevention and/or treatment of genetic diseases (Maeder and Gersbach, 2016). In the last couple of decades, several genome editing tools have been introduced, which include the meganucleases (Epinat et al., 2003), zinc finger nucleases (ZFNs) (Kim et al., 1996), transcription activator-like effector-based nucleases (TALEN) (Christian et al., 2010), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas) (Jinek et al., 2012; Cong et al., 2013; Sander and Joung, 2014). In general, these tools create DNA double stranded breaks (DSB) to trigger genome editing in vivo (Maeder and Gersbach, 2016). The evaluation of the efficiencies and specificities of genome editing tool is essential for their applications and further developments. In our recent published study, we described a reporter that can generate quantitative readout for genome editing efficiency (Kumar et al., 2019). In this system, a ~20 bp target sequence is placed in a multiple cloning site (MCS) which is right after the start codon of Cerulean fluorescence protein (CFP) to generate a frame-shift of the open reading frame (ORF). This frameshifted-CFP (FsCFP) can be used as a reporter of genome editing because only when there is a successful DNA-double strand break (DSB) event on the target sequence followed with a non-homologous end joining (NHEJ) to generate an in/del event to shift the reading frame to a correct order (by a chance of up to 1/3), the CFP fluorescence will be reactivated as a positive readout. To facilitate the quantification, an internal ribosome entry site (IRES) and a red fluorescence protein, mCherryFP, is placed after the reporter. In principle, this reporter can be applied to any genome editing system as long as a DSB and NHEJ are expected from the editing. This approach can effectively detect low-efficiency editing in a population of cells with very low false negative or false positive. Furthermore, in this method, the positive cells can be conveniently identified and enriched for the examination or validation of the in/del event in the genome. Also, this method can be easily adapted for screening to optimize the genome-editing enzyme or the other components (such as guide-RNA) in the positive cells. Here, we used the CRISPR-Cas9 technique as a demonstration and the flow cytometry as the readout of the fluorescence events.

In this protocol, the target sequence is inserted between restriction sites of NotI and XhoI before CFP reporter together with sequence for the optimal recognition of Cas9 and a premature STOP codon to create a frame shift. The reporter region is then integrated in the nuclear genome of the target cell by the assistance of lentivirus. The target cells expressing the red fluorescence protein are then isolated by fluorescence-activated cell sorting (FACS), before vectors containing the Cas9 and gRNA are introduced into these cells. After incubation, the ratio of the CFP over mCherryFP was measured in flow cytometry to provide quantitative measurement for the efficiency of the genome editing.