The Progress of CRISPR/Cas9-Mediated Gene Editing in Generating Mouse/Zebrafish Models of Human Skeletal Diseases

Abstract

Genetic factors play a substantial role in the etiology of skeletal diseases, which involve 1) defects in skeletal development, including intramembranous ossification and endochondral ossification; 2) defects in skeletal metabolism, including late bone growth and bone remodeling; 3) defects in early developmental processes related to skeletal diseases, such as neural crest cell (NCC) and cilia functions; 4) disturbance of the cellular signaling pathways which potentially affect bone growth. Efficient and high-throughput genetic methods have enabled the exploration and verification of disease-causing genes and variants. Animal models including mouse and zebrafish have been extensively used in functional mechanism studies of causal genes and variants. The conventional approaches of generating mutant animal models include spontaneous mutagenesis, random integration, and targeted integration via mouse embryonic stem cells. These approaches are costly and time-consuming. Recent development and application of gene-editing tools, especially the CRISPR/Cas9 system, has significantly accelerated the process of gene-editing in diverse organisms. Here we review both mice and zebrafish models of human skeletal diseases generated by CRISPR/Cas9 system, and their contributions to deciphering the underpins of disease mechanisms.

Coding Variants Coupled With Rapid Modeling in Zebrafish Implicate Dynein Genes, dnaaf1 and zmynd10, as Adolescent Idiopathic Scoliosis Candidate Genes

“…Faithful genetic models of scoliosis, which model aspects of AIS in zebrafish, have been described ( Kusumi and Dunwoodie, 2010 ; Boswell and Ciruna, 2017 ; Wu et al, 2019 ). Thus far, the etiology of scoliosis in these zebrafish models is associated with defects in the development of ependymal cell cilia and abnormal cerebrospinal fluid (CSF) physiology ( Grimes et al, 2016 ; Konjikusic et al, 2018 ), disassembly of the central canal resident glycoprotein structure called the Reissner fiber ( Lu et al, 2020 ; Rose et al, 2020 ; Troutwine et al, 2020 ), and alteration of neuropeptide signaling within the central canal ( Zhang et al, 2018 ; Vesque et al, 2019 ; Lu et al, 2020 ), which altogether appear to contribute to increased neuroinflammation ( Van Gennip et al, 2018 ; Rose et al, 2020 ).…”

New gene discoveries in skeletal diseases with short stature

“…Since 2012, the CRISPR-Cas9 technology ( 78 ) has transformed the field of genetic engineering by introducing the possibility of efficiently silencing a gene of interest (or introducing a specific pathogenic mutation) in commercially available cell lines ( 79 ). While this approach overcomes the problem of obtaining patient-derived osteogenic cells, the use of immortalized cell lines might introduce changes in the cells that might be incorrectly assessed as part of the phenotype induced by the introduced pathogenic variant/gene defect.…”

The Role of Chronic Inflammatory Bone and Joint Disorders in the Pathogenesis and Progression of Alzheimer's Disease

“…The CRISPR-Cas9 system has also been utilized to generate numerous knockout models of inflammatory arthritis and OP. Following the identification of putatively pathological gene segments via genome-wide association studies, these genes of interest are either deactivated, modified, or spliced into model systems (often incorporating iPSCs) to faithfully recapitulate the diseased phenotype in controlled in vitro and in vivo settings—these findings have been thoroughly reviewed elsewhere (Adkar et al, 2017 ; Ding and Orozco, 2019 ; Wu et al, 2019 ; Yuan et al, 2019b ). The simplicity, low cost, and high cleavage efficiency of CRISPR-Cas9 compared to earlier Transcription Activator-Like Effector Nuclease and Zinc Finger Nuclease based systems is appealing; however, its limitations merit serious consideration and currently preclude its use in mainstream clinical settings.…”

“…Firstly, Cas9 is known to generate off-target modifications: a few mismatches distal to the protospacer adjacent motif do not prevent activation of the CRISPR-Cas9 system. Screening alternative Cas orthologs with enhanced specificity and target range has been attempted to mitigate this artifact (Wu et al, 2019 ). Founder mosaicism is another boundary: in knockout and transgenic models of disease, CRISPR-Cas9 components are injected into the fertilized zygote and continuously target and cleave genes during embryonic development, often causing mosaicism in the introduced mutations.…”