This post comes from Fred Nelson, MD, an orthopaedic surgeon in the Department of Orthopedics at Henry Ford Hospital and a clinical associate professor at Wayne State Medical School. Some of Dr. Nelson’s tips go out weekly to more than 3,000 members of the Orthopaedic Research Society (ORS), and all are distributed to more than 30 orthopaedic residency programs. Those not sent to the ORS are periodically reposted in OrthoBuzz with the permission of Dr. Nelson.
Understanding recent gene technology can be very daunting. The CRISPR/Cas9 method for gene editing is a prominent example. CRISPR stands for Clustered Regularly Interspersed Short Palindromic Repeats, and Cas9 is an acronym for the CRISPER-Associated Protein 9. Scientists became aware of CRISPR in E. coli in 1987, but they only recently realized that CRISPR constituted an adaptive immune system for bacteria and archae, which are primitive bacteria-like cells.
When infected by a virus (phage), a bacteria’s Cas genes are activated. Cas gene products cut viral DNA sequence sites called protospacers and then insert those sequences into the bacterial DNA. The host bacterium identifies the viral sequences by a protospacer adjacent motif (PAM), which is rarely seen in the host genome. Hence, replication of this sequence will not adversely affect the host. In the event of a second phage attack, Cas genes are activated and they generate CRISPR RNA (crRNA), which recognizes the phage sequence. crRNA associates with Cas nucleases to cleave both DNA strands of the invader.
There are numerous CRISPR modules. Type II CRISPR is one of an expanding number of naturally existing CRISPR families that has have been used for gene editing in eukaryotes. The type II CRISPR family uses crRNA and an additional tracrRNA to target specific DNA sequences. These have been combined to create a single guide RNA (gRNA) to direct sequence-specific Cas9 double-stranded DNA cleavage. The result is a simple, programmable RNA method that has been used for genome targeting and genome editing in eukaryotes.
The accuracy of this system has been markedly enhanced to avoid unwanted mutations. The system is being fashioned to block existing gene expression, modify gene expression by inserting DNA sequences, and activate expression of single or multiple genes. CRISPR technology enables researchers to develop mouse models of disease much more quickly and less expensively than traditional approaches. Larger animal models of disease can also now be produced.
Successful treatment of mouse models of human diseases with CRISPR suggests that the technology can be applied to directly treat human diseases in the future. Preclinical research is underway using CRISPR-ed stem cells or mouse models to study human diseases such as retinitis pigmentosa, Fanconi anemia, Duchenne muscular dystrophy, sickle-cell anemia, and cystic fibrosis.
Thanks to Dr. Gary Gibson for his help with this tip.
Gibson GJ, Yang M. What rheumatologists need to know about CRISPR/Cas9. Nat Rev Rheumatol. 2017 Apr;13(4):205-216. doi: 10.1038/nrrheum.2017.6. Epub 2017 Feb 9.
Every month, JBJS publishes a Specialty Update—a review of the most pertinent and impactful studies published in the orthopaedic literature during the previous year in 13 subspecialties. Click here for a collection of all OrthoBuzz Specialty Update summaries.
This month, Matthew J. Allen, VetMB, PhD, author of the December 5, 2018 Specialty Update on Musculoskeletal Basic Science, focuses on the five most compelling findings from among the more than 60 noteworthy studies summarized in the article.
Gene Editing in Orthopaedics
–Gene-editing tools such as CRISPR-Cas9 have great potential as a means of introducing therapeutic genes into mesenchymal stem cells that can then be targeted to tissues in vivo. These researchers1 reported on genetically modified stem cells that have the potential to differentiate into chondrocytes encoding a natural inhibitor of interleukin-1, providing an opportunity for localized release of immunomodulatory factors.
Managing Orthopaedic Infections
–A novel study2 in which transmission electron microscopy was used to identify viable bacteria deep within the canalicular structure of cortical bone, remote from the site of an infected implant, suggests that effective debridement requires the removal of not just necrotic tissue, but also of adjacent, apparently unaffected bone.
Computational Modeling of Human Movement
–This report3 presented a human musculoskeletal model that provided extremely accurate predictions of ground reaction forces during simulated walking and squatting. As similar models are developed and validated, surgeons will have improved tools for evaluating patients, planning surgery, and making decisions about which procedure/implant is most appropriate for an individual patient.
–This report4 demonstrated sexually dimorphic regulation of gene-expression profiles in bone marrow osteoprogenitor cells that could partly explain clinical observations in sex differences in peak bone mass, bone remodeling, and immunomodulation.
Biological Enhancement of Ligament Healing
–Among several basic science papers focused on the optimal healing and durable fixation of tendons and ligaments, this notable work5 reported on the translation of bridge-enhanced ligament repair for the anterior cruciate ligament.
- Brunger JM, Zutshi A, Willard VP, Gersbach CA, Guilak F. CRISPR/Cas9 editing of murine induced pluripotent stem cells for engineering inflammation-resistant tissues. Arthritis Rheumatol.2017 May;69(5):1111-21. Epub 2017 Mar 31.
- de Mesy Bentley KL, Trombetta R, Nishitani K, Bello-Irizarry SN, Ninomiya M, Zhang L, Chung HL, McGrath JL, Daiss JL, Awad HA, Kates SL, Schwarz EM. Evidence of Staphylococcus aureus deformation, proliferation, and migration in canaliculi of live cortical bone in murine models of osteomyelitis. J Bone Miner Res.2017 May;32(5):985-90. Epub 2017 Jan 26.
- Jung Y, Koo YJ, Koo S. Simultaneous estimation of ground reaction force and knee contact force during walking and squatting. Int J Precis Eng Manuf.2017;18(9):1263-8.
- Kot A, Zhong ZA, Zhang H, Lay YE, Lane NE, Yao W. Sex dimorphic regulation of osteoprogenitor progesterone in bone stromal cells. J Mol Endocrinol.2017 Nov;59(4):351-63. Epub 2017 Sep 4.
- Perrone GS, Proffen BL, Kiapour AM, Sieker JT, Fleming BC, Murray MM. Bench-to-bedside: bridge-enhanced anterior cruciate ligament repair. J Orthop Res.2017 Dec;35(12):2606-12. Epub 2017 Jul 9.