Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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GENengnews.com | DECEMBER 2017 | 29 Fortunately, genome-editing compo- nents can be loaded into viral vectors, expediting clinical development. An early genome-editing approach used zinc finger nucleases (ZFNs). ZFNs provided proof of concept of genetic repair, and they have been applied in a number of clinical trials. Even more promising is the emergence of CRISPR/Cas9, which represents a new class of genome-editing tools. It can be used to repair the incorrect spelling of a disease-causing gene, and it can do so without leaving behind footprints. Logistical Issues "For research, the CRISPR meth- ods are most convenient, but they have some limitations in the realm of delivery and possibly in the choice of target," comments Dana Carroll, Ph.D., professor of biochemistry, University of Utah School of Medi- cine. "TALENs are the least limited in target selection and have excellent specificity. The ZFNs are the smallest and thus the easiest to incorporate into some viral vectors." "In the private sector, companies can choose among platforms based on IP issues. All platforms are capable of high efficiency and specificity. If a complex project involves only one or a few targets that will be attacked re- peatedly, development of the cleavage reagent will represent a small part of the time and expense of the project, so any of the platforms could be used." With modifications that have been made in the last few years, nearly any sequence can be targeted with adequate specificity, continues Dr. Carroll. Specificity requirements vary among applications, and they need to be evaluated for each new nuclease/ target combination. Delivery of the reagents to the cells or tissues of interest is not a major issue for in vitro and ex vivo applica- tions but remains significant for in vivo applications, both in humans and in model organisms. Lastly, currently indel formation, insertion or deletion of bases, by non-homologous end joining (NHEJ) is more efficient in most cells than sequence replacement or insertion by homologous recombination (HR). Because many applications depend on sequence correction, it would be helpful if HR were more efficient. Consistent- ly better HR efficiency, however, has proven difficult to achieve. "The most obvious clinical appli- cations are ones that can be done ex vivo," states Dr. Carroll. Elaborating, he notes that two approaches are be- ing taken to treating sickle-cell disease by modifying hematopoietic stem cells with CRISPR. One of these approaches is being explored in Dr. Carroll's own laboratory. "The approach that we're studying depends on HR with an oligonucle- otide template to correct the offending single-base-pair disease mutation," Dr. Carroll details. "The other approach uses CRISPR to reverse the repression of fetal beta globin in adults; the per- sistence of fetal hemoglobin is thera- peutic and even curative. "The approved clinical trials that involve CRISPR are simply using it to inactivate genes in therapeutic CAR-T cells in order to enhance their effi- cacy," concludes Dr. Carroll. Inserting Exogenous DNA Creation of knockin and con- ditional knockout animal models requires the insertion of long exog- enous DNA cassettes into specific sites in the genome. The CRISPR/Cas9 system can make a cut at the desired location in the genome, but the cellular DNA re- pair system, NHEJ, immediately seals it back before the homology directed repair (HDR) pathway finds and in- serts the exogenous DNA piece at the cut site. These mechanisms were evaluated in a project led by Masato Ohtsuka, Ph.D., associate professor of mo- lecular life science, Tokai University, and Channabasavaiah Gurumurthy, MVSC, Ph.D., assistant professor of developmental neuroscience, Mun- roe Meyer Institute and director, Transgenic Core Facility, University of Nebraska Medical Center. These scientists demonstrated that if the ex- ogenous DNA is supplied as a single strand, instead of a double strand, the HDR process outpaces NHEJ and efficiently slides in the foreign DNA cassette at the cut site. They termed this strategy Easi-CRISPR ("Easi" refers to B E S T O F C R I S P R 2 017 ➜ Creating one humanized mouse model, using traditional technologies, can take 5–10 years or more; CRISPR-based approaches can shrink the time requirement of genome- engineering experiments several-fold.

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