Genetic Engineering & Biotechnology News

NOV15 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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14 | NOVEMBER 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News Many of these new tools were discussed at Global Technology Commu- nity's 7 th Genomics and Big Data Summit in Coronado, CA, in Sep- tember 2017 and at the CRISPR & Precision Genome Editing conference in Berlin in November 2017. Detecting gene-editing outcomes is of paramount importance. Next-gen- eration sequencing (NGS) is one method; NGS provides all the information needed to know what is happening at a gene locus. But preparation of librar- ies—as well as genome analysis—take time and expertise. Another approach, droplet digital PCR (ddPCR), provides very sen- sitive and precise quantitation of homology-directed repair (HDR), point mutagenesis, and nonhomologous end-joining (NHEJ)—without the need for markers or artificial reporters. The method is relatively quick and inex- pensive compared with NGS. ddPCR and NGS are not mutually exclusive; ddPCR is an alternative for quick turnarounds, especially if a laboratory is focused on a few genes and designing gene-specific probes for deep probing. Chromosomal double-strand breaks trigger DNA repair, and restoration can occur via two cellular pathways: HDR and NHEJ. Although CRISPR/ Cas9 can induce both HDR and NHEJ, NHEJ predominates. HDR is based on recombination between the genomic DNA and the template DNA, and can induce very precise single base-pair resolution editing. And while NHEJ-mediated repair of Cas9-generated breaks is generally effective, it is also prone to errors. 1 CRISPR could be used in more diverse ways if HDR could be induced more efficiently, perhaps, as studies have shown, through the knockout of NHEJ-related factors. 2 Yuichiro (Ichi) Miyaoka, Ph.D., project leader, Regenerative Medicine Program, Tokyo Metropolitan Institute of Medical Science, was the first to use ddPCR to detect CRISPR genome-editing outcomes. His current re- search focuses on finding the best conditions for HDR induction. ddPCR is used to systematically compare different germinating conditions for induc- ing NHEJ and HDR activity, as it can simultaneously detect both events. Many other detection methods are specific for either HDR or NHEJ. New Arrayed Libraries Short interfering RNA (siRNA), used for years for functional genomic screening, has helped to establish best practices paving the way for the CRISPR paradigm. The permanent genome-level edit in CRISPR/Cas9 has its own unique challenges when compared to the transient silencing in RNA interference (RNAi). The same target may be altered in nearby genomic regions, result- ing in unintentional silencing. As opposed to RNAi off-targets, unintentional silencing of one gene sequence may occur, but that gene may be antisense to a noncoding region with regulatory activity, or the two DNA strands may have different roles or the edit may disrupt an intronic noncoding region. Arrayed CRISPR library offerings are relatively new and allow a higher level of interrogation of the biology compared with the limitations of plus/minus in a lentiviral pooled screen. Arrayed libraries of CRISPR RNA (crRNA), such as Edit-R crRNA li- braries (Dharmacon—A Horizon Discovery Company), cover the whole human genome—18,000 OMICS Feature See CRISPR Tools on page 16 CRISPR Tools Grow in Sophistication MaryAnn Labant CRISPR, a tool advancing the field of functional genomics, changed the game with knockouts instead of knockdown, and facilitated more robust phenotyping mapping. As the technology continues to mature, tool suppliers are pulling their weight and offering variations of new guide RNAs (gRNAs) and libraries. Figure 1. crRNA designs assigned high scores by Dharmacon's Edit-R algorithm have higher cleavage efficiency than do crRNA designs assigned low scores. Ten crRNAs with high functional scores for 10 genes (blue) and 10 crRNAs with low functional scores for the same genes (green) were tested for editing by next-generation sequencing. Ninety-three percent of the high-scoring crRNAs and 32% of the low-scoring crRNAs showed >40% of editing (indel formation).

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