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.

Issue link: http://gen.epubxp.com/i/909577

Contents of this Issue

Navigation

Page 68 of 69

GENengnews.com | DECEMBER 2017 | 35 Proxy-CRISPR Experimentation Martha S. Rook, Ph.D., Head of Gene Editing and Novel Modalities, MilliporeSigma M illiporeSigma has a highly active R&D program to explore CRISPR function and practical utility for genome editing. MilliporeSigma, like others, has found that many natural bacterial CRISPR systems cannot func- tion efficiently in human cells to support practical ge- nome editing work. The Cas9 protein from Streptococcus pyogenes (SpCas9), however, works surprisingly well in the chromatinized context of the human genome. Therefore, SpCas9 has become widely adopted, and MilliporeSigma chose it to assume the role of "hypoth- esized chromatin disruptor" in its initial proxy-CRISPR experiments. MilliporeSigma made a mutant of SpCas9 that lacks DNA cutting activity (dead-SpCas9 or dSpCas9) and targeted it to adjacent locations of less active CRISPR systems (such as FnCpf1, FnCas9, CjCas9, etc.). To the company's surprise, proximal targeting of dSpCas9 had a strong and consistent positive effect on these various bacterial CRISPR systems in human cells. Proxy-CRISPR can enhance genome editing projects in at least three important ways: (1) it can boost genome editing activity, including both gene knockout and knockin; (2) it can enable targeting of DNA cutting closer to desired mutation sites, which exponentially in- creases knockin activity; and (3) it creates a requirement for two binding events to achieve knockout/knockin, thus reducing the frequency of off-target cutting. Enabling Protein Biology Studies in a More Natural Biologic Context Amy Landreman, Ph.D., Global Product Manager, Promega T ag sequences are often incorporated into expressed proteins facilitating characterization of various aspects of pro- tein function. However, traditional over- expression models can result in experimental artifacts by removing proteins from the native genomic context and changing stoichiometry with interacting partners. Endogenous gene tagging with CRISPR allows pro- tein studies under physiologically relevant conditions. However, the large size of many tags results in low inser- tion efficiency, and detection is often restricted to anti- body-based methods that limit throughput, sensitivity, and the ability to obtain quantifiable results. Promega scientists have overcome these limitations by combining an 11-amino-acid peptide tag with sensitive bioluminescent detection, HiBiT, and CRISPR editing. We find efficient insertion of the HiBiT tag by using a single-stranded oligodeoxynucleotide donor molecule, which is ordered directly, eliminating the need for molecular cloning. HiBiT is detected using a simple add-and-read biolu- minescent method that has over 7 logs of linear dynam- ic range. Such sensitivity enables quantification of HiBiT- tagged proteins in pools of edited cells, rendering the process from editing to assay as quick as 24–48 hours. We have used endogenous HiBiT tagging to study the regulated abundance of proteins HIF1A and BRD4 and found that expression from the endogenous lo- cus results in significantly improved assay response compared to transient overexpression. Our work has demonstrated the HiBiT tag to be highly amenable to CRISPR knockin, enabling protein biology studies in a more natural biologic context. A CRISPR Way of Modifying Animals Abhi Saharia, Ph.D., Director of Product Management, Synthego C RISPR/Cas9 has transformed the generation of modified animals by enabling gene modifications in a multitude of previously intractable species. The CRISPR toolbox has itself evolved rapidly with novel syn- thetic technologies that have further accelerated and increased the probability of project success. High-efficiency CRISPR reagents such as chemically modified synthetic single-guide RNA (sgRNA) from Synthego have set a new high bar for successful ge- nome editing. Data from the laboratory of Joseph M. Miano, Ph.D., associate director of the Aab Cardiovacular Institute at the University of Rochester Medical Center, demonstrates that delivering sgRNA and Cas9 protein (as ribonucleoproteins or RNPs) to the one-cell animal em- bryo via microinjection has resulted in the rapid produc- tion of new genetic animal models that previously would be considered risky or even challenging to engineer. This optimized approach to generating and genotyp- ing "CRISPRized" animals has led to a nearly 100% suc- cess rate of germline transmission in the animal models, enabling rapid generation of genetically modified ani- mals in the span of weeks with almost a guarantee of success. This level of success has led Dr. Miano to com- ment on how well Synthego's sgRNAs work the mouse zygote: "Not only has Synthego saved us valuable time, we are getting higher efficiency precision editing by HDR. We have completely switched over to Synthego's sgRNA and Cas9 protein for RNP injections, and we are not looking back." n B E S T O F C R I S P R 2 017

Articles in this issue

Links on this page

Archives of this issue

view archives of Genetic Engineering & Biotechnology News - DEC 2017