Applications of CRISPR for musculoskeletal research

Figures

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  Fig. 1. Basic outline of Cas9 genome editing involving the two major DNA repair pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). The initial step is for the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 complex to bind to its DNA target. The CRISPR-Cas9 complex comprises Cas9 endonuclease (yellow) and single guide RNA (gRNA) and RNA scaffold (green) that binds to a specific DNA dictated by the gRNA. DNA cleavage will only occur when the gRNA is adjacent to the protospacer adjacent motif (PAM) recognition sequence. Following double-strand cleavage by the Cas9 endonuclease, DNA repair can be mediated by one of two possible mechanisms: NHEJ or HDR. Repair by NHEJ (left pathway) leads to the inclusion of DNA insertions or deletions (orange) resulting in a reading-frame shift, leading to a premature stop codon in the downstream sequence and the ‘knocking-out’ out of the targeted allele. Repair by HDR (right pathway) in the presence of donor DNA (red) targeted to the cut site results in insertion of the donor DNA sequence at the site of the DNA strand break and a ‘knock-in’ allele. Image adapted from Cribbs AP, Perera SMW. Science and…

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  Fig. 2. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The CRISPR-Cas9 system and its ability to target specific genomic loci has been adapted for a range of new applications. These include improvements to the Cas9 nuclease itself (A), active or inactive Cas9 fused to different protein modules for enhanced functionality (B, D, and E), and Cas isoforms that have high affinity for RNA instead of DNA (C). These advances have been applied to all the musculoskeletal tissues including cartilage, bone, and muscle.