Genetic Engineering & Biotechnology News

DEC 2017

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20 | DECEMBER 2017 | GENengnews.com Dr. Behlke. Moreover, IDT created chemically modified derivatives of the two components of the guide RNA to reduce the risk of triggering the innate immune system. This highly optimized RNP com- plex could be delivered by any stan- dard transfection technique. Microin- jection of Alt-R ® CRISPR/Cas9 RNP system is particularly valuable for generation of knockout and transgenic mice. Electroporation of primary T cells with Alt-R RNP complex target- ed to alpha chain of the T-cell receptor routinely results in over 80% knock- out of surface TCR complex, without detectable toxicity (Figure 1). "Our next significant improvement is a novel Cas9 mutant," continues Dr. Behlke. Using directed evolution, sci- entists developed a high-fidelity Cas9 mutant that fully retains on-target activity with significantly reduced off- target activity. The Alt-R RNP com- plex—combining chemically modified synthetic guide RNA and the high- fidelity Cas9 mutant—brings CRISPR technology even closer to clinical ap- plications. Using I DT reagents, a team of Stanford University scientists led by Matthew Porteus, M.D., Ph.D., dem- onstrated robust results in correcting sickle-cell mutations in bone marrow stem cells, potentiating translation into clinical trials. Such developments, suggests Dr. Behlke, should justify an optimistic outlook: "Precision editing of [the] genome for human health is not such a distant reality." Zebrafish Genome Explains Hearing Loss The human sense that most com- monly becomes impaired is hearing. In the United States alone, reductions or losses in the ability to detect sounds have been experienced by approxi- mately 28 million people. When hearing becomes impaired, the underlying cause is usually (more than 50% of the time) genetic. Various genetic causes of hearing impairment are of interest to Lisa A. Schimmenti, M.D., a medical geneticist at the Mayo Clinic Rochester. For example, she investigates ge- netic variations that affect a protein called gap junction beta-2 (GJB2). These variations account for more than 50% of hearing loss cases world- wide that are nonsyndromic (i.e., not associated with other symptoms), and for about 25% of such cases among children in North America. Families carrying GJB2 variants demonstrate considerable spread in phenotype from completely deaf to mild impair- ment, suggesting other contributing factors. To model human hearing, Dr. Schimmenti's laboratory relies on the Using CRISPR to Improve Disease Modelin g Continued from page 18 B E S T O F C R I S P R 2 017 The CRISPR/Cas9 system has greatly enhanced gene editing in eukaryotic organisms. However, there is a continued need to im- prove both cleavage activity and guide RNA design density and specificity. In the exploration of CRISPR systems, MilliporeSigma sci- entists report that they have observed type II-B FnCas9 from Francisella novicida to possess a higher intrinsic specificity than SpCas9. Motivated by its high specificity, the investigators set out to examine the robustness of FnCas9 in comparison with SpCas9. In contrast to SpCas9, FnCas9 activity fluctuated greatly along several genomic regions and failed to cleave a large number of the targets in chromosomal regions at which SpCas9 cleaved efficiently, notes Martha Rook, Ph.D., head of gene editing and novel modalities at MilliporeSigma, who adds that regional vari- ability in endonuclease activity is not exclusive to FnCas9. "We also observe type II-C CjCas9 and NcCas9 and type V FnCpf1 to display little or no cleavage at an array of genomic loci," she points out. "Several studies suggest that chromatin structures can be a major barrier to RNA-guided endonuclease binding and cleavage in mammalian cells." After observing consistent activity on purified DNA sub- strates and testing variations in guide RNA length at cleavable and apparently uncleavable genomic sites, the team reasoned that target-site accessibility might be the limiting factor in edit- ing efficiency in these cases. "Our scientists hypothesized that the binding of an orthogonal, catalytically dead S. pyogenes Cas9 (SpdCas9) at proximal loca- tions might alter local chromatin structures and render other- wise inaccessible target sites now accessible and subsequently cleavable by alternative nucleases such as those mentioned above," continues Dr. Rook. "In a recent publication [Chen, et al., "Targeted Activation of Diverse CRISPR/Cas Systems for Mammalian Genome Editing via Proximal CRISPR Targeting," Nat. Commun. 8, 14958 (2017)], we show a restoration of alternative nuclease activity by co-target- ing SpdCas9 to bind at proximal sites. Our scientists indicate that this proxy-CRISPR targeting strategy is applicable to a range of genomic loci and as a proof of concept, shows that the strategy may enable selective editing between identical genomic sites by judiciously selecting proximal dCas9-binding sites to differenti- ate the identical genomic locations." These findings provide a method to improve both genome- editing precision and efficiency using diverse CRISPR systems and a path to a better understanding of the impact of chromatin microenvironments on genome modification, states Dr. Rook. n Examining FnCas9

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