Guan, Y et. al. EMBO Mol Med (2016) http://www.ncbi.nlm.nih.gov/pubmed/26964564
Hemophilia B is a genetic blood clotting disorder that results from a mutation in coagulator factor IX (FIX) normally treated through intravenous deliver of FIX. As an increase in FIX levels by as little as 1% can lead to significant clotting improvement, Hemophilia B is uniquely suited for gene therapy treatment. Guan et al. utilized the CRISPR/Cas9 system and homology-directed repair to correct the mutation in the FIX gene. While the HDR rate was found to be 0.56% efficient with a single-stranded DNA donor and 1.5% efficient with a double-stranded plasmid donor, the rate of repair was great enough to restore hemostasis.
31Jocelyn Kaiser, Science Magazine, December 31st, 2015. http://news.sciencemag.org/biology/2015/12/crispr-helps-heal-mice-muscular-dystrophy
Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy that primarily affects boys and usually results in death around age 25. DMD is the result of defects or missing DNA in 79 exons that make up the dystrophin gene. Due to the large size of the dystrophin genes, traditional gene therapy approaches have not been able to fully replace the faulty dystrophin gene. In the December 2015 edition of Science, three independent groups report using CRISPR/Cas9 technology to remove defective exons of the dystrophin gene in young mice resulting in a truncated form of dystrophin (http://www.ncbi.nlm.nih.gov/pubmed/26721686, http://www.ncbi.nlm.nih.gov/pubmed/26721684, http://www.ncbi.nlm.nih.gov/pubmed/26721683). While the treated mice showed considerable improvement compared to the controls, the treatment cannot be considered a cure since they did not perform as well on muscle tests as normal mice. Despite its shortcomings, this technique 31could improve the quality of life for those living with DMD.
Christopher Alessi and Jonathan Rockoff, The Wall Street Journal, December 20th, 2015. http://www.wsj.com/articles/bayer-in-venture-with-gene-editing-startup-1450644397
Germany based Bayer AG has partnered with CRISPR Therapeutics AG to develop gene editing therapies for conditions such as hemophilia, infant heart disease, and Stargardt blindness. CRISPR Therapeutics was founded by Dr. Emmanuelle Charpentier, co-author of the breakthrough 2012 CRISPR Science paper with Dr. Jennifer Doudna. The deal is worth $300 million over five years with Bayer acquiring a $35 million stake in CRISPR Therapeutics.
Gantz V.M. and Ethan Bier. Science (2015) 348:442-444. http://www.ncbi.nlm.nih.gov/pubmed/25908821
Zoonotic diseases, such as malaria and West Nile, result in illness and can be fatal. Traditionally these diseases have been controlled by individuals wearing insect netting and by reduction in insect populations. With the advent of gene editing techniques such as CRISPR/Cas9 systems so called “Gene Drives” have become a third possibility. Gene Drives involve the selective gene editing and propagation throughout a population to prevent carrier infection. In order for a Gene Drive to be successful both copies of a gene must be edited in the modified individual to ensure transmission to all offspring. Gantz and Bier have now demonstrated the ability to use the CRISPR/Cas9 system to edit both alleles in somatic and germline cells through a process called mutagenic chain reaction (MCR). In Drosophila this involves insertion of the CRISPR/Cas9 machinery through Homology Directed Repair into the target genome which allows for continuous editing of the target site through insertion of the CRISPR/Cas9 machinery.
Schwank, G. Cell Stem Cell (2013) 13:653-658. http://www.ncbi.nlm.nih.gov/pubmed/24315439
CRISPR/Cas9 systems hold promise as a therapeutic gene therapy for a variety of chronic genetic diseases. Cystic Fibrosis is the result of mutations in both alleles of the CTFR gene and is currently not a treatable condition. In this study the authors present results demonstrating the ability of a CRISPR/Cas9 system to replace the mutant copy with a functional allele through homologous-end-joining repair in cultured small intestine cells of a cystic fibrosis patient. The specificity of CRISPR/Cas9 systems could allow for gene therapies to cure genetic diseases.
Sternber, S.H. et al. Nature (2015) 527:110-113. http://www.ncbi.nlm.nih.gov/pubmed/26524520
One of the major obstacles facing CRISPR/Cas9 systems is off-target cleavage events. Understanding the mechanisms by which CRISPR/Cas9 systems identify and cleave DNA could provide insights into ways to decrease off-target cleavage. While Cas9 crystal structures have been elucidated the HNH domain active site was found to be ~30Å away from the targeted DNA thus preventing a detailed understanding of the cleavage mechanism. By developing a Förster resonance energy transfer (FRET) based system Sternber et al were able to determine that two α-helices induce a conformational shift that acts as a signal transducer to activate both the HNH and RuvC nuclease domains thus coordinating cleavage of both DNA strands. Furthermore, the authors determined that Cas9 could cleave off-target sites that contained only 1-3 mismatches a the distal end of the guide RNA since the activating conformation shift was still able to occur.
Kleinstiver, B.P. et al, Nature (2015) 523:481-485. http://www.ncbi.nlm.nih.gov/pubmed/26098369
While the construction of unique guide RNAs theoretically allows CRISPR/Cas9 systems to target any unique genomic sequence the range of possible sequences is limited by the need for a specific protospacer adjacent motif (PAM). For instance, the widely adopted Cas9 endonuclease requires an NGG PAM to directly follow the guide RNA sequence. To overcome this limitation Kleinstiver et al engineered Cas9 variants to recognize novel PAM sequences using a directed evolution approach thus expanding the range of sequences CRISPR/Cas9 systems can be used to edit.
Nicholas Wade, The New York Times, December 3rd, 2015. http://www.nytimes.com/2015/12/04/science/crispr-cas9-human-genome-editing-moratorium.html
An international panel called for a pause in using CRISPR/Cas9 technology to alter genes that could be inherited by future generations. This moratorium was the result of a recent international meeting held at the National Academy of Sciences in Washington, jointly called by the National Academy of Sciences, the Chinese Academy of Sciences, and the Royal Society of London in response to the use of CRISPR/Cas9 technology to alter a non-viable human embryo earlier in 2015. While the moratorium does not have regulatory authority, it is expected most scientists will follow the new guidelines. The respective scientific bodies did leave the door open for removal of the moratorium, stating the issue “should be revisited on a regular basis.”
News Office, MIT, December 1, 2015. http://news.mit.edu/2015/overcome-crispr-cas9-genome-editing-hurdle-1201
One of the major obstacles facing wider adoption of CRISPR/Cas9 systems as a gene editing and gene therapy technique is the off target cleavage of DNA. In a report published in Science (http://www.sciencemag.org/content/early/2015/11/30/science.aad5227.abstract) MIT researchers used rational protein engineering to improve CRISPR/Cas9 specificity. Using the crystal structure of the CRISPR/Cas9 complex researchers identified a positively charged groove that is able to stabilize binding of negatively charged DNA. By changing three of the positive amino acids found in this groove to neutral amino acids the researchers reasoned that DNA binding would become more dependent on proper Watson-Crick base pairing between the CRISPR guide RNA and the targeted DNA. This improved Cas9, coined “enhanced specificity” SpCas9 or eSpCas9, eliminated 22 of the 24 off-target events observed when compared to wild-type Cas9 treatment.
Amy Harmon, New York Times, November 26th, 2015. http://www.nytimes.com/2015/11/27/us/2015-11-27-us-animal-gene-editing.html
Gene editing technology, such as CRISPR/Cas9 and TALEN systems provide the opportunity to edit not only the plants humans consume but animals as well. Gene editing technology is currently being applied to the cattle industry to create hornless dairy cows in order to prevent farmers from needing to dehorn their livestock. More immediately consumers will have access to salmon that has been modified in order to substantially increase its growth rate. Gene editing is also being used for therapeutic goals with pigs being altered to grow human organs that will not be rejected and to make malaria resistance mosquitoes all in an effort to improve human health. While these technologies provide many opportunities some scientist and activists are promoting caution while the FDA has yet to release guidelines for gene editing. In order for gene editing technology to become widely adopted scientist, the public, and government regulators will need to come to a consensus as to the extent to which this technology is used.