**8. Limitations and Future Considerations**

The CRISPR/Cas9 system is being used in a wide range of applications and many studies. However, notwithstanding its meteoric rise in a short period of time and its potential applications in medicine and beyond, it, like other genome-editing tools, does come with limitations and concerns, ethical and otherwise. One of these limitations is the effective targeting range, as the sgRNA can only bind to a region near a specific PAM sequence on the DNA. The PAM sequence for Cas9 is 5 -NGG-3 , where "N" can be any nucleotide base, but the third base must be G. This can greatly reduce the potential target locations available to make DNA edits such as insertions or deletions. In experiments conducted by Nishimasu and colleagues, a Cas9 with a more relaxed preference for the PAM third base resulted in the recognition of an NGD PAM instead of an NGG PAM requirement. This effectively increased the potential targets for Cas9 nuclease, as the NGD sequence occurs more frequently in human DNA than NGG sequences. The engineered Cas9 in this instance had a wider range and increased cleavage specificity, reducing instances of off-target incisions. The new Cas, termed SpCas9-NG, can bind to A, G, or T in the 3rd base of the PAM sequence [118].

MicroRNAs (miRNAs), small, non-coding RNA molecules, may help to regulate inflammation, promote MSC differentiation, and ensure the homeostasis of cartilage [119]. As a key factor in epigenetic regulation, miRNAs can change the gene expression without modifying the sequence of the DNA that encodes proteins [120]. This may be a much safer way to modulate gene expression since the genome sequence does not change, and the gene expression pattern may still be inheritable from one cell generation to the next [85]. miRNAs may be used in combination with CRISPR/Cas9 and EVs to design patient-specific approaches to the treatment of OA [103].
