Programmable Molecular Scissors: Applications of a New Tool for Genome Editing in Biotech

Abstract

Targeted genome editing is an advanced technique that enables precise modification of the nucleic acid sequences in a genome. Genome editing is typically performed using tools, such as molecular scissors, to cut a defined location in a specific gene. Genome editing has impacted various fields of biotechnology, such as agriculture; biopharmaceutical production; studies on the structure, regulation, and function of the genome; and the creation of transgenic organisms and cell lines. Although genome editing is used frequently, it has several limitations. Here, we provide an overview of well-studied genome-editing nucleases, including single-stranded oligodeoxynucleotides (ssODNs), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and CRISPR-Cas9 RNA-guided nucleases (CRISPR-Cas9). To this end, we describe the progress toward editable nuclease-based therapies and discuss the minimization of off-target mutagenesis. Future prospects of this challenging scientific field are also discussed.

Keywords: CRISPR-Cas9; DSB; HDR; NHEJ; TALENs; ZFNs; genome editing; nucleases; off-target mutagenesis; ssODNs.

Figure 1

Genome Engineering Using Programmable Nucleases Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 are used to induce targeted double-strand breaks (DSBs) at the desired chromosomal locus. Non-homologous end joining (NHEJ) or homology-directed repair (HDR), one of the two cellular repair pathways, is then used to repair the DSB. NHEJ can be used to knock out genes, whereas HDR can be used either for gene correction or to introduce precise alterations into the genome; this is directed by a homologous DNA template. Adapted from Chandrasegaran and Carroll and Ramalingam et al., copyright (2015) Creative Commons Attribution 4.0 International.

Figure 2

An Overview of Applications of Genome Editing Genome editing can be used in various fields of biotechnology, including biopharmaceutical development and gene therapy; genome structure alteration, regulation, and function; and production of biofuel, food, and transgenic cell lines and animals.

Figure 3

Illustration of DNA Recognition by ZFPs and Crystal Structures of FokI and How FokI Bound to DNA (A) DNA recognition by ZFPs. (Ai) Structure of a single ZFN, (Aii) DNA recognition by ZFNs, and (Aiii) structure of three-finger Zif268 bound to its cognate site. (B) Crystal structures of FokI and FokI bound to DNA. (Bi) Structures of FokI-DNA complex and of FokI enzyme alone. In both structures, the FokI cleavage domain piggybacks on the recognition domain. (Bii) Native FokI crystallizes as a dimer. The dimer interface is at the FokI nuclease domain, which is formed by two salt bridges between arginine (R) and aspartic acid (D) residues of the FokI monomers. Reproduced from Chandrasegaran and Carroll, Wah et al., Miller et al., and Pavletich et al., copyright (2015) Creative Commons Attribution 4.0 International.

Figure 4

An Overview of Potential Applications of Programmable Nucleases Programmable nucleases can edit the genome and reprogram genetic information, which consequently affects genome structure and function. This technology can be used to produce farm animals, transgenic cell lines (i.e., embryos, stem cells, and induced pluripotent stem cells [iPSCs]), and transgenic plants.

Figure 5

Schematic Diagram of Potential Therapeutic Applications of Genome Editing

Figure 6

A Schematic Diagram for Production of Genetically Engineered Mouse Models Using Programmable Nuclease (CRISPR-Cas9) Genetically engineered mouse models (GEMMs) can be used to discover mechanisms of drug resistance and those of tumor initiation and progression, and to develop new anticancer drugs.

Figure 7

Schematic Illustration of the Use of Induced Pluripotent Stem Cells in Relation to Alzheimer’s Disease (A) Induced pluripotent stem cells (iPSCs), derived from a skin biopsy acquired from a patient with Alzheimer’s disease (AD), are differentiated into neural progenitor cells and neurons. (B) In familial cases, the disease-causing mutation can be corrected by gene editing of the iPSCs; the neural progenitor cells and neurons can be used for research and drug screening. (C) Patients can benefit from cell therapy, better diagnostic procedures, customized treatments, and novel medical approaches. Reproduced from Freude et al., copyright (2014) Creative Commons Attribution 4.0 International.

Figure 8

Schematic Illustration Showing Functional Repair of CFTR by CRISPR-Cas9 in Intestinal Stem Cell Organoids Acquired from Patients with Cystic Fibrosis Reproduced from Schwank et al. Copyright 2013. Reproduced with permission from Elsevier.