Precision gene editing technology and applications in nephrology

Abstract

The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision. Progress has also been made in harnessing endogenous DNA repair machineries, such as non-homologous end joining, homology-directed repair and microhomology-mediated end joining, to functionally manipulate genetic sequences. As understanding of how DNA damage results in deletions, insertions and modifications increases, the genome becomes more predictably mutable. DNA-binding platforms such as TALEs and CRISPR can also be used to make locus-specific epigenetic changes and to transcriptionally enhance or suppress genes. Although many challenges remain, the application of precision gene editing technology in the field of nephrology has enabled the generation of new animal models of disease as well as advances in the development of novel therapeutic approaches such as gene therapy and xenotransplantation.

Figure 1.. Commonly Used Programmable DNA Platforms:

A diagram showing programmable DNA binding platforms that recognize double-stranded DNA (dsDNA). A. A pair of 4-Finger Zinc Finger proteins binding to each side of the desired double-stranded break (DSB) location in the DNA. Each Zinc Finger (ZF) domain binds three bases of DNA; multiple ZF domains can be stringed together to bind longer stretches of DNA. When bound, the attached FokI nuclease (N) dimers become close enough in proximity to activate and catalyze a double stranded DNA break. B. A pair of Transcription Activator Like Effector domains (TALEs) bound to each side of the preferred DSB position. TALE domains consist of a series of 35 amino acid repeats attached in sequence. Each of these motifs binds to a single specific DNA base and can be strung together to recognize diverse DNA sequences. C. The CRISPR/Cas9 endonuclease system functions through the interaction of a RNA guide with a single protein, Cas9. The RNA guide consists of two domains, a constant poly-hairpin structure that interacts with the Cas9 protein and a programmable guide region that targets DNA through standard Watson-Crick base pairing. Upon binding its target region by interrogating and unwinding (melting) the dsDNA, the Cas9 protein induces a blunt double stranded break. D. Another CRISPR system makes use of different class of guide RNA coupled with a different constant protein, Cas12a. Targeting is again determined by Watson-Crick base pairing between the guide RNA and the DNA, following which Cas12a induces a DSB with its signature overhang.

Figure 2.. Associated Biochemical Activities Critical For Precision Gene Editing:

Diagram showing the different cellular repair machineries and corresponding biochemical functions critical for gene editing. A. Repair of a DSB by Non-Homologous End Joining (NHEJ) introduces mutation through the creation of small insertions or deletions (indels). B. NHEJ repair can delete long segments of DNA (whole genes) by creating two double stranded breaks and removing the intervening region from the chromosome C. Homologous Recombination (HR) can be used to insert exogenous DNA though the introduction of an exogenous template flanked by large (often >500 base pairs) dsDNA sequences homologous the sequence adjacent to the DSB. D. Oligo-directed HDR can introduce small changes through the introduction of a short single-strand DNA (ssDNA) template flanked with homology matching the regions to either side of the DSB. E. Micro-Homology Mediated End Joining can be used to create small reproducible deletions. This repair pathway functions by annealing small homologous regions on each side of the DSB. Unbound DNA flaps are removed and ends are ligated resulting in the removal of one homology arm and the intervening region. F. MMEJ can also be used to insert exogenous DNA through the introduction of a template with matching microhomology arms. G. Single nucleotide polymorphisms can be introduced without a DSB using a base editor. By targeting a cytosine deaminase to a specific target in the genome it is possible to convert a cytosine to a uracil resulting in a mismatch base. This mismatch is then recognized and repaired, generating a single base change depending on which strand is chosen as repair template, this can be selected by design by repeated nicking of the strand to be modified.

Figure 3.. Precision Epigenetic Modulation

A. Programmable methylation of CpG islands in DNA can be achieved by fusion of any of the DNA binding platforms described in this review to a sequence non-specific methyltranferase (MTase) or Trans-eleven Translocation enzyme (TeT) to methylate or demethylate the DNA, respectively B. Fusion of a DNA binding system to histone acetyltransferases (HATs) or histone deacetylases (HDACs) enables programmable acetylation or deacetylation of specific lysine residues of the histone proteins associated with the target DNA. C. Artificial transcription factors can be created by the fusion of transcription activating or suppressing domains to any DNA binding system. When bound to promoter or enhancer regions, transcription levels of genes can be modulated without any chemical modification to the DNA or associated proteins.

Figure 4.. Exponential Reduction in Cost of Gene Editing Tools and Subsequent Rapid Growth of Deployment in Scientific Publications.

Left axis – chart shows representative commercial costs of gene editing tools from ZFNs in 2011 to TALENs in 2013 and CRISPR-Cas9 in 2016. Right axis -chart shows the number of publications in PubMed using each indicated gene editing platform since 1995. The exponential reduction in cost and the greatly increased access to these new tools is reminiscent of the Moore’s Law of continual reduced cost underlying computer technology.