The applications and advances of CRISPR-Cas9 in medical research

Guest Editors: Lu Xiao-Jie and Ji Li-Juan, Xiao-Jie Lu, Hong-Mei Sun, Yong Xu, Xi Yu, Biao Gu, The applications and advances of CRISPR-Cas9 in medical research, Briefings in Functional Genomics, Volume 16, Issue 1, January 2017, Pages 1–3, https://doi.org/10.1093/bfgp/elw036

Since its introduction into mammalian cells [ 1, 2] and animals [ 3] in 2013, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) has revolutionized many fields of medical research, including disease modeling [ 4], therapeutic explorations [ 5], genetic screens [ 6], etc. CRISPR-Cas9 is a RNA-guided genome editing tool derived from a microbial adaptive immune defense system [ 1, 2, 7]. It mainly comprises a nuclease termed Cas9 and a single-guide RNA (sgRNA) that is complementary to the target sequence. On the presence of a protospacer-adjacent motif on the opposite strand, sgRNA recognizes the target strand via base pairing and thus guides Cas9 to bind to and cut target DNA sequence [ 8]. The resultant DNA double-strand breaks are repaired by either nonhomologous end joining (NHEJ) or homologous-directed repair (HDR) [ 1, 2]. NHEJ is error-prone in that it may generate unpredictable indel mutations, whereas HDR can produce desired gene replacement [ 9, 10]. The charm of CRISPR-Cas9 lies in its multiplexing nature and ease of use. CRISPR-Cas9 is multiplexing in that multiple loci can be targeted simultaneously if provided multiple sgRNAs [ 2]. CRISPR-Cas9 is easy to use because retargeting requires only the redesign of a sgRNA, which is much easier than the de novo synthesis of a bulking guiding protein as required in conventional programmable nucleases such as zinc finger nuclease and transcription activator-like effector nuclease.

CRISPR-Cas9 has many variants that can function beyond genome editing. Catalytically, dead Cas9 (dCas9), for example, can function as a RNA-guided DNA-binding domain that when fused to different effectors can exert different functions in a site-specific manner, such as live imaging of genome loci of interest [ 11] when fused to fluorescent proteins and epigenetic modulations when fused to epigenetic modifiers [ 12, 13]. Besides transporting functional effectors directly by dCas9, the 3′ end of sgRNA can also be extended to form a RNA scaffold capable of recruiting RNA-binding proteins that are fused to functional effectors. In recent years, CRISPR-Cas9, along with its various variants, has been widely applied to medical research for diverse purposes.

One of the early applications of CRISPR-Cas9 in medical research is disease modeling. Duchenne muscular dystrophy (DMD), for example, is caused by mutations in the DMD gene encoding dystrophin. By simultaneously targeting two exons in the rat DMD gene with CRISPR-Cas9, Nakamura and colleagues [ 14] abrogated dystrophin expression in rats and achieved a rat model of DMD. Moreover, CRISPR-Cas9 can model multigenic diseases more conveniently and faithfully than conventional transgenic techniques. Cancer, for example, is a disease rooting in multiple genetic mutations. Cancer modeling with conventional transgenic techniques often necessitates laborious and time-consuming processes of germ line manipulation and animal cross-breeding, and it is hard to faithfully recapitulate the complexity of oncogenic mutations. CRISPR-Cas9, on the contrary, can directly induce multiple somatic mutations in adult mice [ 15, 16], bypassing the needs of germ line manipulation and animal cross-breeding. Moreover, the multiplexing nature of CRISPR-Cas9 enables modeling of multiple oncogenic mutations simultaneously or sequentially. Xue and colleagues [ 15], for example, delivered CRISPR-Cas9 components into adult mice through tail vein hydrodynamic injection and successfully induced both gain- and loss-of-function (LOF) mutations in mouse liver cells that promoted hepatocarcinogenesis. To model the oncogenic Eml4–Alk gene inversion, Maddalo et al. [ 16] delivered CRISPR-Cas9 components targeting both Alk and Eml4 genes into mouse lungs and gained a mouse model of lung adenocarcinomas with properties and phenotypes similar to those of ALK + human lung cancer.

Besides disease modeling, CRISPR-Cas9 has also been applied to functional genomic screens. There are two fundamentals for the application of CRISPR-Cas9 in functional genomic screens: the availability of mass synthesis of sgRNA library through oligonucleotide synthesis technology [ 17], and the ability of the CRISPR-Cas9 system to edit multiple genomic loci simultaneously [ 2]. Owing to their versatility in genomic and epigenome editing, CRISPR-Cas9 and its variants enable multiple screen formats such as LOF screens [ 18, 19], activation screens [ 12, 13] and knockdown screens [ 12]. These screen formats can be used for multiple purposes such as identification of genes involved in resistance to adverse conditions (drugs or toxins) [ 18] and interrogation of gene functions in health and diseases [ 19].

The most amazing area in the applications of CRISPR-Cas9 is its potential for gene therapy. Just several months after its introduction into mammalian cells [ 1, 2], CRISPR-Cas9 demonstrated its potential in gene therapy by mutating HIV-1 to decrease its expression in human T cells [ 20]. Since then, much effort has been made to explore the therapeutic potentials of CRISPR-Cas9 in combating infections such as hepatitis B virus [ 21] and human papillomavirus [ 22], in correcting culprit mutations in monogenic diseases in model organisms [ 23, 24] and in inducing therapeutic or protective mutations in host cells [ 25–27]. Recently, important progresses have been made in this regard. Three studies [ 28–30] published simultaneously in Science, for example, reported that CRISPR-Cas9 components delivered through intramuscular, intraperitoneal or intravenous injection corrected the culprit gene mutation in mouse models of DMD and rescued the disease phenotype. Two studies [ 31, 32] published back to back in Nature Biotechnology

demonstrated the efficacy of CRISPR-Cas9-mediated HDR for in vivo gene therapy through intravenous injection in mouse models of human hereditary liver diseases. Compared with previous studies in cell lines or animal germ lines [ 20–27], these five recent studies [ 28–32] represent a significant step forward because they have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy delivered by methods that are potentially translatable to human use [ 28–32].

In company with these advances and applications are ethic and safety concerns. When the first study [ 33] reporting the engineering of human reproductive cells with CRISPR-Cas9 was published in 2015, profound concerns had been raided across the scientific community and beyond [ 34–36]. Undoubtedly, there remains a big uncertainty on what CRISPR-Cas9 will bring to us if it is applied to modify the human genome, given that it can introduce permanent and inheritable changes to our genome and that it may incur unpredictable and uncontrollable off-target effects. Therefore, an international consensus is urgently needed to put genome engineering technologies under regulation to ensure the safety and ethicality of their usage.

In this special issue, we included six review articles from distinguished researchers in this field. We discussed the applications and advances of CRISPR-Cas9 in medical research in areas such as disease modeling, therapeutic explorations and genomic screens, and we also pay attention to the ethical issues of this technology and discuss the possible risks and benefits this technology will bring to us. In the three articles by Kato etal. [ 37], Torres-Ruiz etal. [ 38] and Zuckermann etal. [ 39], the authors discussed the applications of CRISPR-Cas9 to in vitro and in vivo modeling of human diseases. They not only provided technique details but also discussed challenges and proposed possible solutions. In the article by Qi etal. [ 40], the authors discussed the applications of CRISPR-Cas9-mediated genomic screens in functional identification and investigation of genes in human health and diseases. Koo and Kim [ 41] discussed in their article, the potentials of CRISPR-Cas9 in human gene therapy and raised concerns and possible solutions on this issue, whereas the article by Ishii dwelt on the ethical issues of CRISPR-Cas9 [ 42]. We are honored if this special issue can raise awareness of this novel technology and can stimulate future studies on this regard.

Funding

This work was supported by the National Natural Science Foundation of China (81472242, 81570549), Shanghai Municipal Health Bureau Key Disciplines Grant(ZK2015A24), National Basic Research Program Grant (2014CB943104), Natural Science Foundation of the Science and Technology Commission of Shanghai Municipality (14ZR1431600, 14411973700), and Shanghai Municipal Health Bureau (20134100).