Updated summary of genome editing technology in human cultured cells linked to human genetics studies

Abstract

Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

Regulating genetic engineering guided by human dignity, not genetic essentialism

“…Consider several examples: Some analysts regard gene editing as a “reliable molecular toolbox” (Bayat et al, 2018, p. 107) to “precisely alter genomes for numerous applications” (Batzir et al, 2017): from basic research to clinical application, and from developing “animal models for genetic disorders” to “gene therapy to combat virus infectious diseases,” and even to “correct monogenic disorders in vivo or in pluripotent cells” (Huang et al, 2017, p. 3875). Germline genome editing in human embryos can program cells “for diverse applications, including regenerative medicine and cancer immunotherapy” (Ho & Chen, 2017, p. 57). It can prevent parents’ giving serious genetic diseases to their offspring (Ishii, 2017, p. 418). It can correct “mutations in patient cells,” and unique gene therapies can screen out causative mutations and identify “rare genetic disorders and non-exonic mutation-caused diseases” (Miyamoto et al, 2018, p. 133). It can enhance the “efficacy of genome editing in the early embryo” and enable the “generation of allele types previously incompatible with in vivo mutagenesis” (Mianné et al, 2017, p. 68). Personalized, molecular surgeries on “genetic DNA directly target the cause of the disease in a personalized and possibly permanent manner”; they “could be combined with traditional surgery, radiation therapy, or chemo/targeted therapy” (Tang & Schrager, 2016, p. 83). By “replacing the mutation-carrying mitochondria of zygotes or oocytes at risk with donated unaffected counterparts,” germline genome editing in human embryos may prevent a “broad range of incurable inborn maladies” caused by mutant mitochondrial DNA (Adashi & Cohen, 2018). While “no curative treatment for patients with mitochondrial disease” exists, germline gene replacement therapy (unlike prenatal and preimplantation diagnosis) may someday prevent transmission of mitochondrial disease (Amato et al, 2014).…”

“…It can prevent parents’ giving serious genetic diseases to their offspring (Ishii, 2017, p. 418). It can correct “mutations in patient cells,” and unique gene therapies can screen out causative mutations and identify “rare genetic disorders and non-exonic mutation-caused diseases” (Miyamoto et al, 2018, p. 133).…”

The precise magic of CRISPR

“…Not all cell types can be readily subjected to HDR‐mediated gene editing due to their differential repair and proliferative capacities [9]. For example, primary cells are restricted in their proliferation potential, making it hard to obtain sufficient genetically identical material from a single edited cell.…”

Disruptive Technology: CRISPR/Cas-Based Tools and Approaches

“…CRISPR/Cas-type molecules thus have great potential both as therapeutic tools and for the identification of future therapeutic targets. Combined with the wider importance of CRISPR/Cas technology for therapy development, where it also contributes to several-fold accelerated development of disease models by facilitating the creation of isogenic cell-line [136–138] and animal [138–146] models, CRISPR/Cas technology has already become an essential and ubiquitous component of biomedical research for infectious, malignant and nonmalignant diseases.…”