A New World Order: Tailored Gene Targeting and Regulation Using CRISPR (2014)

Not since the discovery of RNA interference has a new technology made such a powerful impact in the molecular biosciences. The RNA-guided, modified type II prokaryotic clustered regularly interspaced palindromic repeats system (CRISPR) represents not only an exceptional tool but also potentially an important new therapeutic modality. CRISPR, in its native function, provides adaptive immunity in bacteria by introducing targeted DNA mutations in pathogenic viruses and plasmids (reviewed in ref. 1Mali P Esvelt KM Church GM Cas9 as a versatile tool for engineering biology.Nat Methods. 2013; 10: 957-963Crossref PubMed Scopus (892) Google Scholar). The breakthrough technology came in 2012 with the development of modified CRISPR components that worked in human cells. This simplified system was found to be easily adapted into expression vectors to target any DNA sequence from virtually any organism, thereby greatly expanding its function and utility. To date this system has been used in human cells to target gene excision, activating and repressing proteins, as well as to instill gene fusions. CRISPR has the advantage over other gene excision approaches, such as those based on transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), in that a small guide-RNA component is easily programmable and remains physically separate from Cas9/dCas9 expression. CRISPR/Cas9 might prove to be a useful future therapeutic option for the targeting, editing, or inactivation of disease gene sequences, whether inherited or acquired. Two impressive studies point to powerful therapeutic possibilities for this technology. Schwank and colleagues applied CRISPR/Cas9 to target and correct the CFTR locus in cultured intestinal stem cells of cystic fibrosis patients.2Schwank G Koo BK Sasselli V Dekkers JF Heo I Demircan T et al.Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.Cell Stem Cell. 2013; 13: 653-658Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar The corrected allele was shown to be fully functional in clonally expanded intestinal organoids. In a second study, a dominant inherited disease was corrected in zygotes of mice carrying mutations in a cataract-causing gene, Crygc.3Wu Y Liang D Wang Y Bai M Tang W Bao S et al.Correction of a genetic disease in mouse via use of CRISPR-Cas9.Cell Stem Cell. 2013; 13: 659-662Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar Few question the value of CRISPR/Cas9 as a genomic editing tool. However, its therapeutic potential is less certain—at least, at this time. Two overriding concerns are immediately apparent. First, it is unclear whether expression of this nonhuman protein will be tolerated. For example, expression of exogenous marker proteins such as green fluorescent protein can elicit substantial unwanted immunological responses.4Stripecke R Carmen Villacres M Skelton D Satake N Halene S Kohn D Immune response to green fluorescent protein: implications for gene therapy.Gene Ther. 1999; 6: 1305-1312.Crossref PubMed Scopus (295) Google Scholar Second, despite efforts aimed at reducing off-target effects, previous studies with endonucleases have shown that even minor nonspecific cleavage of DNA can result in chromosomal deletions and rearrangements as well as cellular damage. Moreover, there is the possibility of targeting closely related gene sequences. Although efforts should be made to address these concerns, contingencies are already available that can address safety-related issues for therapeutic applications. For example, a “hit and run” approach may prove beneficial where exposure to CRISPR/Cas9 is highly restricted or where cells can be extensively screened ex vivo before their therapeutic use. An example of this option is the targeting of HIV-1 infection. Proof-of-concept studies have already used CRISPR/Cas9 to induce targeted deletions of the HIV-1 co-receptor CCR5 (refs. 5Cradick TJ Fine EJ Antico CJ Bao G CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity.Nucleic Acids Res. 2013; 41: 9584-9592.Crossref PubMed Scopus (467) Google Scholar, 6Cho SW Kim S Kim Y Kweon J Kim HS Bae S et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141.Crossref PubMed Scopus (1002) Google Scholar) as well as the integrated HIV-1 promoter (ref. 7Ebina H Misawa N Kanemura Y Koyanagi Y Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus.Sci Rep. 2013; 3: 2510.Crossref PubMed Scopus (408) Google Scholar). The modified CRISPR/Cas9 system is undoubtedly a powerful and far-reaching tool for biology and medicine. A wide range of functions has been identified, and one can envision many more potential functions in which guide RNAs recruit factors to a specific genomic address. Clearly, the therapeutic application of CRISPR/Cas9 is still in its infancy, but with the astonishing pace of discovery one can only wonder what the next 18 months have to offer.