![]() In all cases of leukemia, the SCID X-1 correcting gene had inserted into the patient genome within or near tumor-promoting genes and caused transcriptional activation (Check, 2002 Kaiser, 2003 Thomas et al., 2003). However, five children subsequently developed T-cell leukemia, with one child dying from chemotherapy-refractory leukemia. Of the 20 patents participating in the trial, 17 were successfully and stably cured (Cavazzana et al., 2016). Perhaps the most well-known early gene therapy trial involved two studies from France (Hacein-Bey-Abina et al., 2002 Hacein-Bey-Abina et al., 2010) and the UK (Gaspar et al., 2004 Gaspar et al., 2011) of children suffering from X-linked severe combined immunodeficiency (SCID X-1). Early efforts to correct disease-causing genetic mutations in humans, although generally successful, were tainted by several tragedies. To date, over 3000 genes have been associated with disease-causing mutations (Cox et al., 2015). Gene therapy may greatly benefit from CRISPR/Cas9 technology. This broad impact of the CRISPR/Cas9 gene editing tool has led to over 6000 research publications since its development five years ago. However, the simplicity and specificity with which CRISPR/Cas9 can edit DNA is changing the pace of biological research in many areas, including identifying and understanding mechanisms of genetic diseases (Findlay et al., 2014 Gilbert et al., 2014 Zhou et al., 2014 Konermann et al., 2015), validating disease targets (Shalem et al., 2014 Wang et al., 2014), developing animal disease models (Wang et al., 2013 Yang et al., 2013), facilitating genetic engineering in plants (Raitskin & Patron, 2016 Zhang et al., 2016, 2017), and allowing for more thorough epigenetic studies (Yao et al., 2015 Vora et al., 2016). Much of this enthusiasm centers on the clinical potential of CRISPR/Cas9 for treating human disease and editing the human genome. ![]() The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.ĭiscovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) (Ishino et al., 1987 Mojica et al., 1993 van Soolingen et al., 1993), their function as part of an adaptive prokaryotic immune system (CRISPR-associated system, Cas) (Bolotin et al., 2005 Mojica et al., 2005 Pourcel et al., 2005 van der Oost et al., 2009), and subsequent development into a genomic editing tool (Jinek et al., 2012 Cho et al., 2013 Cong et al., 2013 Mali et al., 2013), has revolutionized the field of molecular biology. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. liposomes polyplexes gold particles), and discuss their relative merits. adeno-associated virus (AAV) full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. microinjection electroporation), viral delivery methods (e.g. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. ![]() The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. ![]() In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. ![]() Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Gene therapy has long held promise to correct a variety of human diseases and defects. ![]()
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