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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30 | DECEMBER 2017 | efficient additions with ssDNA in- serts). Easi-CRISPR addresses a major challenge of animal genome engineer- ing. Whereas dsDNAs serve as poor donors for insertions at cut sites, the efficiency of ssDNA insertions in Easi- CRISPR has been demonstrated to be 25–100%. Over a dozen different models with 1,000–1,500 base pairs have been generated. In another project, Drs. Ohtsuka and Gurumurthy worked with Masa- hiro Sato, Ph.D., professor of molecu- lar biology, Kagoshima University, to develop genome-editing via oviductal nucleic acids delivery (GONAD). In GONAD, DNA and CRISPR reagents are introduced into a female mouse oviduct via electroporation, eliminat- ing the traditional gene-editing steps of isolating fertilized eggs, microinjecting them with DNA, and then transferring the embryo into a new set of females. As technology evolves further, the molecular mechanisms of the HDR process needs to be better understood to enable large-scale genome engineer- ing, wherein clusters of human genes can be inserted en masse to create humanized animal models. Creat- ing 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. Tracking Editing CRISPR consists of two compo- nents: a guide RNA (gRNA) and a nonspecific CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a sequence necessary for Cas9 binding and a custom sequence that defines the genomic target to be modified. Standard gel-based assays, such as T7E1, CEL-I, and Surveyor, can be used to determine gRNA activity. These assays, which use the enzyme mismatch cleavage method, are not very accurate; however, they are easy to adopt in any laboratory. Another qualitative method is PCR amplification of the region containing the modification site followed by Sanger sequencing. Multiple peaks will result if double- strand break repair has occurred by aberrant rejoining, which indicates gRNA activity. These peaks can be deconvoluted if the Sanger traces are subjected to additional analysis. For example, the traces can be evaluated according to the TIDE (Tracking of Indels by DEcomposition) method. Editing results can also be checked with a method called targeted deep sequencing. It is not typically imple- mented in individual laboratories, due to instrument and reagent costs, as well as difficulties related to data analysis. It is, however, a sensitive and quantitative approach. "Targeted deep sequencing has changed our lives," exclaims Shondra Miller, Ph.D., director, Genome Engineering and iPSC Cen- ter (GEiC), Washington University School of Medicine. "We can tell at a very sensitive level how much and what type of editing is occurring. Next-generation sequencing (NGS) gives a true picture of what is hap- pening in a population of cells or even in a single-cell-derived clone." NGS provides a lot of informa- tion quickly, and is used for identify- ing knockouts and for determining knockout or knockin rates. Reagents Genome Editing Explores New Depths Continued from page 29 B E S T O F C R I S P R 2 017 The Genome Engineering and iPSC Center (GEiC), a resource established by the Washington University School of Medicine, facilitates functional genomic studies through the use of patient-derived induced pluripotent stem cells and the generation of modified cells and organisms using genome-editing technologies. The GEiC serves both the academic and private sectors.

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