Alt-R CRISPR Genome Editing: Innovation Driving Solutions for HDR, Specificity, and Off-Target Analysis
Integrated DNA Technologies, Inc.
Genome editing by CRISPR systems has proven to be groundbreaking in medical research and has great implications for the treatment of human disease. CRISPR-Cas9 and Cas12a (Cpf1) proteins introduce double-stranded breaks (DSBs) at targeted genomic loci, which are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). CRISPR systems demonstrate unparalleled editing efficiency in a broad range of host species and cell types but suffer from concerns related to target site specificity and editing precision.
Here, we describe methods for detecting and measuring the frequency of off-target effects (OTEs) including multiplexed enrichment for next-generation sequencing and data analysis to fully characterize on- and off-target editing events. In addition, we present novel mutant CRISPR enzymes with improved on- and off-target editing profiles: Alt-R HiFi S.p. Cas9 nuclease and Alt-R A.s. Cas12a Ultra nuclease. Finally, we describe methods to improve the rate of HDR-mediated repair by careful selection of repair template characteristics and the use of Alt-R HDR Enhancer. We implement these findings in the Alt-R CRISPR HDR Design Tool, a novel bioinformatics tool for ssDNA HDR template design.