Gene Editing: Powerful New Tools for Nephrology Research and Therapy

Abstract

Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.

Keywords: gene therapy; molecular genetics; transgenic mouse.

Figure 1.

Simplified schematic illustrating DSB repair mechanisms induced by CRISPR/Cas9. The three pathways, HDR, NHEJ, and MMEJ, are used for different gene editing purposes. The black triangles indicate locations of the cut site. NHEJ leads to frequent small insertions or deletions that can lead to disruptive frameshift mutations and premature stop codons. This approach is best suited for generating point mutants or knocking out a gene. Both HDR and MMEJ pathways can be used to introduce longer DNA sequences, but they each require an exogenous donor DNA template. These mechanisms are well suited for introducing precise mutations or knocking in an epitope tag or reporter gene, such as green fluorescent protein. Modified from Sakuma et al., with permission.

Figure 2.

Schematic illustration of a gene knockout using CRISPR/Cas9 gene editing. (A) Two gRNAs were designed on the coding start region and 3′ untranslated region (UTR), respectively, of gene of interest (GOI) to cut out about 9 kb. The light blue rectangles and green rectangles show coding sequences and UTRs, respectively. The screening primers are located outside of the gRNAs. (B) PCR is performed on DNA isolated from individual clones of cells subject to gene editing. The extension time is purposefully brief, so that the 9-kb WT allele cannot be amplified. Different sizes of deleted alleles are detected, because each clone has different deletion lengths near the DSB.

Figure 3.

Illustration of a C–terminal P2A-eGFP–floxed puromycin expression cassette knock-in for gene of interest (GOI) by CRISPR/Cas9 editing. (A) A gRNA was designed to overlap with the stop codon of a GOI, and the donor vector contained homology arms of 800–1000 bp. After HDR, a P2A-eGFP–floxed puromycin expression cassette will be inserted in frame just after the coding region and before the stop codon. Screening primer 1 was designed outside of the left homology arm to prevent detection of the donor plasmid. The insert–specific primer 2 was designed on eGFP. (B) Results of pooled genomic PCR from mouse embryonic stem cells that detects the insertion of the desired sequence into the genome. (C) Sequence results from a single clone indicate precise integration of the desired sequence into the genome. Only the results of the 5′ sequence are shown. UTR, untranslated region.