Enhancing efficiency and specificity with improved CRISPR components and a new CRISPR analysis pipeline

Genome editing with an expanded CRISPR-Cas tool set increases the opportunity to make intentional, targeted changes in the genome. Furthermore, improved specificity of genome editing and ability to fully characterize and quantify these events is a centerpiece of preclinical studies. The CRISPR Research and Development team at IDT remains focused on delivering reagents, tools, and methods to allow scientists to optimize the performance of genome editing workflows. In this webinar, I will focus on optimized parameters to obtain high rates of homology-directed repair (HDR), which allows the generation of specific mutations or insertions in the genome. This includes editing methods with both Cas9 and Cas12a proteins and strategies to introduce short insertions or SNPs as well as large insertions or knock-ins. There will be an interactive demonstration introducing our new NGS data analysis tool for CRISPR applications: CRISPAltRations. The presentation of our cloud-hosted data analysis tool will lead into a discussion of a workflow for complete characterization of on- and off-target editing efficiency and specificity. In addition, I will discuss the expanded genome editing space afforded by the dramatically improved activity of A.s. Cas12a Ultra and L.b Cas12a Ultra. These engineered Cas12a mutant proteins present advantages for genome editing in mammalian cells and plants. We will also introduce fluorescently tagged Cas proteins that allow for visualization in uptake experiments and maintain potent editing activity. Finally, I will introduce expanded gRNA manufacturing capabilities that maintain quality and rapid turnaround times and allow for genome engineering work in high throughput experiments or in animal models that demand more material made possible with large-scale synthesis capabilities. Additionally, our new custom gRNA ordering interface will allow for easy design and ordering of a gRNA to suit any targeted Cas nuclease of interest from a diversity of species.

Learning Objectives:

1. Our latest developments and customizations for guide RNAs, including pegRNAs and CRISPR libraries, and our newest CRISPR nucleases with fluorescently labeled Cas9 variants

2. Improved design tools, enhancing reagents, and analysis tools used to efficiently generate SNPs and large knock-ins

3. Simplified methods for high-throughput detection and analysis of CRISPR-induced off-target effects, including a demonstration of our new, biologically informed CRISPR NGS analysis pipeline, CRISPAltRations