**2. Current Treatment Options and Limitations**

The progressive erosion of articular cartilage is a prevalent symptom of OA [35]. Articular cartilage is made up of chondrocytes embedded within a collagenous extracellular matrix (ECM) [36]. Disruption of the articular cartilage prevents pain-free movement and affects the load-bearing abilities of the joint. Current treatments for OA can include intra-articular joint injections of steroids and arthroscopic lavage with debridement; however, these may have short-term benefits without long-term benefit to the cartilage [22,37,38]. Anti-inflammatory and analgesic drugs may help to address some of the symptoms of OA, but they do not tackle the fundamental pathologies involved in the inexorable degenerative process, in addition to having significant side effects associated with prolonged use [39–42]. Surgery and joint replacement, if possible in the early stages of OA, are expensive and may need to be performed multiple times during an individual's life [22,43]. Alternative modalities of treatment may well be helpful in the short term, but may not be a sustainable strategy for OA treatment and potential prevention [44,45].

The best way to combat the root cause of joint degeneration in OA may be through the exploration of the various possibilities that genome editing offers [46,47]. There is increasing evidence that genetic and epigenetic modifications play a substantive role in OA; however, it has been difficult to separate the individual effects from the combined effects of genetic and environmental factors acting together to cause progressive joint degeneration [48–51]. Characterizing and analyzing the genetic factors underpinning the pathology of OA may provide viable options for the diagnosis, prognosis, and development of novel treatment targets for future personalized biological therapies.

#### **3. CRISPR**/**Cas**

Several gene therapy systems such as viral, engineered scaffold, and other approaches hold promise [52]. However, while such systems hold promise, the advantages are offset by some potential disadvantages. Here we focus on the CRISPR/Cas9 system, a very powerful gene therapy tool.

CRISPR/Cas is a novel, versatile, and promising genome-editing technique that is opening up new avenues and possibilities in the effective treatment of OA [53] and other degenerative joint diseases [54,55]. CRISPR is an acronym for "clustered regularly interspaced short palindromic repeats", discovered through investigation of the prokaryotic adaptive immune system. It was identified as an effective system used by bacteria and archaea to remember infecting agents such as phage viruses and destroy them upon subsequent exposure, similarly to the memory cells in the human immune system. When paired with different proteins, specifically enzymes such as Cas, that are produced naturally by the prokaryotic cells, the CRISPR system can be used to make deletions, insertions, substitutions, or other changes at specific sites of the prokaryotic and eukaryotic genome [56,57]. From finding a cure for cancer, to treating sickle-cell disease, to growing drought- and pest-resistant crops, CRISPR has many exciting possibilities and potential in several fields [58].

The prokaryotic CRISPR/Cas system has three main components: a Cas nuclease, a crRNA (CRISPR RNA), and a tracrRNA (trans-activating crRNA). In bacterial cells, the CRISPR/Cas system works by recognizing the invading bacteriophage DNA, chopping it up into several pieces, and incorporating them into its DNA. These CRISPR pieces are then transcribed, generating a crRNA and a tracrRNA to create a double-stranded RNA structure that can recruit the Cas proteins. When the offending phage is encountered again, the CRISPR/Cas system is directed to a specific location on the foreign DNA because of a protospacer adjacent motif (PAM) short nucleotide base sequence which is upstream of the crRNA targeted sequence. The Cas protein is programmed to be the "molecular scissors" of the system which carries out the cutting, splicing, and any other editing that is desired [59–61]. The therapeutic application of CRISPR/Cas is illustrated in Figure 2.

**Figure 2.** CRISPR/Cas mechanism. Trans-activating RNA (orange) with CRISPR RNA (blue) the guide RNA. The guide RNA assembles with the Cas9 protein to form the CRISPR complex. Using the guide RNA for specificity, the CRISPR complex binds to the target DNA. Transgenic DNA can be inserted using homology arm inserts.

The most widely used of the six major types of CRISPR/Cas system, the Type II system, is derived from the *Streptococcus pyogenes* bacteria. It uses the Cas9 protein because of its wide working range and efficiency [62]. The CRISPR/Cas9 system that is used currently by researchers and scientists worldwide consists of a single guide RNA (sgRNA) with a binding end (analogous to tracrRNA) for the Cas9 nuclease to attach and a targeting end (similar to crRNA) with nucleotide base pairs that are complementary to the DNA sequence that is meant to be edited (Figure 2). Only the targeting end of the sgRNA needs to be synthesized for any specific targeting sequence, while the binding end does not need to be redesigned every time, thus reducing the time needed to get the tool ready for editing. Cas9 recruitment to the exact DNA target sequence is mediated by the sgRNA. Cas9-induced double-stranded breaks (DSBs) are repaired either by spontaneously nonhomologous end joining (NHEJ) or by homology-directed repair (HDR) using a synthetic donor DNA template [63]. CRISPR/Cas RNA-guided DNA endonuclease genome targeting is much easier to design and apply when compared to other available site-specific editing tools using engineered nucleases such as transcription activator-like effector nuclease (TALENS) and zinc finger nucleases (ZFNs), which are controlled by protein–DNA interactions. CRISPR/Cas is also much more cost- and time-effective because researchers only need to code for a small section of sgRNA [64].

#### **4. Mesenchymal Stem Cells and Tissue Regeneration**

Tissue regeneration and self-renewal of articular cartilage, in general, is a very limited process [65]. The avascularity of articular cartilage may hinder progenitor cells access to the site of injured cartilage [66]). It may also limit molecular factors that are vital to extracellular matrix repair and homeostasis [67–70].

Chondrocytes originate from mesenchymal stem cells [71,72]. Bone-marrow-derived MSCs (BM-MSCs) have much promise in aiding articular cartilage repair due to their proximity to the joint, high differentiation capability, and ability to secrete different growth, anti-inflammatory, and immunomodulatory factors [73–77]. They could affect a clinically relevant improvement in joint pain and function. Additional studies are needed, however, to demonstrate the efficacy of cultured

versus noncultured BM-MSCs and the best ways to deliver them into the joint. MSCs derived from fetal cells also have therapeutic properties, but ethical concerns have been raised about using them for treatment and therapeutic applications [78,79]. Additional challenges arise due to the potential of fetal MSCs to differentiate into several different types of cells, which might be more difficult to control and direct [76,80,81]. The capabilities of MSCs are usually age-dependent. MSCs have a short lifespan but can secrete paracrine factors that may be beneficial in tissue regeneration [81,82]. In addition to BM-MSCs, MSCs derived from other sites such as adipose tissue can be isolated, expanded, characterized, and used to regenerate cartilage [83,84]. However, MSCs tend to form mechanically inferior fibrocartilage instead of the glassy, hyaline cartilage that covers the ends of bones at articulating joints [85].
