CRISPR-Cas9 genome editing is a technology that allows scientists to alter the genome of any organism. This technology is moving towards therapeutic research applications and has been employed in gene and cell therapy trials. CRISPR-Cas9 genome editing is a fast, efficient technique that cuts double-stranded DNA, modifying gene function, but genome editing is not always precise. After DNA is edited, any off-target edit locations must be nominated and fully characterized in order to understand the specificity of the editing event. The rhAmpSeq amplicon sequencing system is a multiplexed, PCR-based targeted next generation sequencing approach that can accurately quantify editing efficiency and specificity.
rhAmpSeq amplicon sequencing quantifies genome editing quickly and efficiently by simultaneously sequencing on- and off-target edits. Garrett Rettig, PhD, the Director of Product Development in the Molecular Genetics Research department at Integrated DNA Technologies (IDT), demonstrates how off-target editing sites are first nominated using GUIDE-seq and studied in detail using the rhAmpSeq system. He provides an overview of rhAmp™ PCR using the rhAmpSeq system, a technology developed at IDT that allows specific detection of editing events that result from CRISPR-mediated double-stranded breaks in genomic DNA. He reviews the features and performance of the rhAmpSeq system, highlighting the flexible assay design and fast workflow. Dr Rettig shows how rhAmpSeq technology can be combined with Alt-R S.p. HiFi Cas9 Nuclease V3 RNP delivery to validate a 99% on-target editing rate using a high-throughput workflow for library preparation. CRISPRAltRations, IDT’s data analysis pipeline, processes these results directly from a sequencing run on an Illumina instrument for fast and easy feedback of editing results.
Dr Rafet Basar, MD, from the lab of Dr. Katy Rezvani at the MD Anderson Cancer Center, presents a case study of this genome editing workflow in virus-specific, cytotoxic T cells. Their team silenced the glucocorticoid receptor via CRISPR-Cas9 editing to reduce its lymphocytotoxic effects, enabling safe, effective treatment of viral infections following hematopoietic stem cell transplantation in immunocompromised patients. They collaborated with IDT scientists to characterize and quantify the off-target effects of their CRISPR experiment and were able to minimize those effects using Alt-R Hifi Cas9. Their work, presented here, demonstrates the applicability of the Alt-R and rhAmpSeq workflows to studies that require large-scale, GMP-compliant throughput.
Watch the recorded webinar below to learn more about the workflow and how it can improve your CRISPR experiments.