On & Off-Target Genome Editing Techniques
Advances in genome editing tools and techniques featured as one of the main topics of conversation during Oxford Global’s annual NextGen Omics 2022 event. Held in London on the 9th and 10th of November 2022, the 2-day congress brought together some of Europe's most successful omics research experts to discuss the latest therapeutic innovation, applications, and technologies within the field of genome editing.
Among the most well-received talks was given by Richard Stanton, Professor of Virology at Cardiff University, who spoke on the possibility of high-throughput cloning. This article looks at the key takeaways from his presentation.
An Optimised CRISPR/Cas9 Adenovirus Vector (AdZ-CRISPR) For High Throughput Cloning Of sgRNA, Using Enhanced sgRNA & Cas9 Variants
By: Richard Stanton, Professor of Virology at Cardiff University
The AdZ-CRISPR vector system provides a simple and efficient way to deliver CRISPR/ Cas9 machinery to cells in vitro and in vivo. Adenovirus (Ad) vectors are viruses with a 36kb double-stranded DNA genome; they are non-enveloped viruses and are easy to convert into a vector by deletion of the E1 region.
The “AdZ-CRISPR enables simple, rapid, and high throughput cloning of sgRNA spacers into an Ad vector.”
Traditionally, the process of making an adenovirus vaccine is relatively laborious, the reason for this being the genome’s large size. “We wanted to streamline and update the process by bringing a wide range of new technologies to the adenovirus cloning platform,” Richard Stanton, Professor of Virology at Cardiff University, explained.
The laboratory experimented with the techniques: applying recombineering to adenovirus vectors, making the vector self-excising, and repressing transgene expression in packaging cells. From this, the laboratory was able to establish the AdZ vector system, with the ‘Z’ referring to the zero cloning steps involved. Stanton describes the system as a “rapid and high-throughput workflow for producing adenovirus vectors expressing transgenes of interest”.
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More recently, the laboratory identified a need to upgrade the AdZ vector system for CRISPR/Cas9 purposes. The “AdZ-CRISPR enables simple, rapid, and high throughput cloning of sgRNA spacers into an Ad vector.” Within this workflow, vectors are maxiprepped and transfected, with the addition of modified Cas9 enzymes such as SniperCas9 supporting more accurate editing without the loss of on-target activity.
Stanton concluded by explaining how modifying sgRNA structures enhances editing efficiency and supports high-efficiency editing of hard-to-transfect cells in vitro and in vivo.
The Conversation Continues at NextGen Omics US 2023...
The upcoming NextGen Omics US 2023 in-person symposium is scheduled for 30th and 31st March and will be held in Boston.
It is a comprehensive 2-day meeting that will discuss gene editing advances, and how key players in academia and pharma are developing techniques to improve accuracy, create more realistic models of disease, and run ground-breaking clinical trials.
Visit our Omics Portal to learn more about the latest research into spatial omics and its developments. If you’d like to register your interest in Oxford Global‘s upcoming NextGen Omics US event, click here.
