CRISPR/Cas9 is a relatively new tool in molecular biology with an enormous variety of potential applications in genomic editing. CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats”, and Cas9 is an associated protein that can induce double-stranded breaks (DSBs) in DNA. CRISPR itself is a form of adaptive immunity found in specific archaea and bacteria. In Type II CRISPR, “invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity” .
The genomic editing applications of CRISPR/Cas9 utilize the same mechanisms as the CRISPR system in bacteria. A single guide RNA (sgRNA) can be designed to direct Cas9 to a particular sequence of DNA next to a specific 2-5 nucleotide sequence called the Protospacer Associated Motif (PAM). Specifically, this process takes advantage of how Cas9 induce DSBs in DNA . When combined in vitro, a DSB is generated at the specified site, and a DNA repair mechanism called Non-Homologous End Joining (NHEJ) is activated. Since NHEJ is error-prone, the repaired DNA may contain insertions or deletions (commonly referred to as “indels”), which can alter the desired gene .
CRISPR/Cas9 is not simply limited to gene knockdown, however. Through the use of a deactivated form of Cas9 (dCas9), gene silencing and gene activation are also enabled . Yet dCas9 has no endonuclease activity, meaning it does not generate DSBs at its target site. However, it can still bind to DNA, and depending on the site where dCas9 binds, it can either largely hinder transcription from occurring (gene silencing) or activate and promote transcription. DNA can also be visualized through the use of dCas9 fused to a fluorescent protein.
In 2015, a research team found that CRISPR/Cas9 “efficiently mutate and deactivates HIV-1 proviral DNA in latently infected Jurkat cells” . The researchers also found that “HIV-1 gene expression and virus production were significantly diminished” . These findings mention just one exciting potential role of CRISPR/Cas9 in retroviral therapies at the genome level.
The genomic editing applications of CRISPR/Cas9 seem only restrained by potential off-target effects. Since “Cas9 can tolerate up to five base mismatches within the protospacer region” , there is potential for unwanted mutations to occur. This could be highly dangerous especially if these mutations occur in critical genes. Although off-target effects using CRISPR/Cas9 can be minimized , they still present an obstable to using CRISPR/Cas9 genomic editing technology in vivo.
Cas9 and generally other programmable endonucleases are very promising for the future of biomedical research, especially since they possess the potential for personalized therapies, as seen in the example of therapy for HIV . Moving forward, however, minimizing off-target effects is a critical step towards making CRISPR/Cas9 an even more effective tool at treating disease at the genomic level.
 New England Biolabs. “CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology.” 2014. Retrieved from https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology
 Zhu W, Lei R, Duff YL, Li J, Guo F, Wainberg MA and Liang C, (2015) The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology 12:22. doi:10.1186/s12977-015-0150-z