Optimizing generation of human isogenic iPSC lines via CRISPR prime editing

Authors

• SF Syed
• PG Robey
• K Futrega

Abstract

CRISPR prime editing (PE) enables precise and versatile genomic modification without inducing double-stranded breaks. PE requires multiple components, including a prime editor enzyme complex, pegRNA (finds the target and provides the new sequence), and sgRNA (directs prime editor complex to nick non-edited strand). Optimization of PE components is essential for most genomic targets. Here, we use PE to introduce type 2 diabetes (T2D) candidate risk variants into human induced pluripotent stem cells (iPSCs), a cell type which can be differentiated into numerous disease-relevant cell types. We tested various PE component systems and developed a highly efficient pipeline that generated isogenic lines carrying heterozygous or homozygous risk or non-risk alleles for six T2D loci.
We found that PE can support editing in iPSCs, but optimization of all components is critical to achieve high efficiency. PE systems utilizing PEmax, epegRNA modifications, and MLH1dn provide significant benefit, with maximal editing efficiency ranging from 36-73%. Editing success for each variant differs considerably depending on the sequence at the target site. pegRNA design also plays a critical role, as slight variations in sequence guide parameters can have significant effects. Established guidelines for pegRNA design should be followed during the initial optimization for each target before making small changes to pegRNA lengths and/or shifting its position to enhance editing rate. Although considerable effort is required to achieve acceptable efficiencies for PE, it is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.

Scientific Focus Area: Genetics and Genomics

This page was last updated on Monday, September 25, 2023