Marker-free co-selection for successive rounds of prime editing in human cells

Abstract

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.

Nucleotide metabolism constrains prime editing in hematopoietic stem and progenitor cells

“…This level of product purity contrasted with the high level of tandem duplications observed at ATP1A1 with the PE3 strategy in the presence or absence of dNs and Vpx VLPs ( Supplementary Fig. 3 ), corroborating previous findings in K562 cells with pegRNA and nick sgRNA PAMs facing outward towards each other (PAM-out configuration) 11 .…”

“…Considering the lower product purity observed when using an additional nick sgRNA, we next tested whether modulating the nucleotide metabolism could positively interact with MMR evasion to achieve high levels of editing with the PE2 approach. A simple yet very efficient approach to evade MMR is to install additional silent mutations near the intended edit 2,19 , as previously demonstrated with ATP1A1 -Q118R v1-v3 epegRNAs 11 ( Fig. 2a ).…”

“…We first tested whether delivering Vpx via virus-like particle (VLP) transduction could enhance prime editing. We cultured HSPCs in the presence of cytokines for 24 hours, and electroporated cells with a first-generation prime editor (PE2) mRNA and a previously optimized synthetic epegRNA/nick sgRNA pair targeting ATP1A1 11 . Following electroporation, we cultured HSPCs for 72 hours in the presence or absence of Vpx VLPs.…”

“…4b , c ). Of relevance, we noted that the highest level of improvement was observed with the ATP1A1 -Q118R_v2 epegRNA, which was optimized to evade DNA mismatch repair (MMR) 11 while the HBB and B2M epegRNAs introduce a single nucleotide substitution and are thus sensitive to MMR. Since MMR antagonizes prime editing efficiency and fidelity 2,16 , we hypothesized that modulating the nucleotide metabolism could positively interact with MMR evasion to facilitate prime editing in HSPCs.…”

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

“…Optimization of the prime editor proteins resulted in the most advanced PE4max and PE5max 179 , while the editing efficiency can be further increased by synonymous mutations in pegRNA 181 or engineered pegRNAs to stabilize the RNA Review article https://doi.org/10.1038/s41467-023-35886-6 structure 179,[181][182][183][184][185] . Other optimizations of prime editing include adding the Rad51 DNA-binding domain 186 , optimized NLS composition 187 , coselection with puromycin 188 , fluorescent reporter 189,190 or cellular resistance 191 , peptide fusion 192 , transient inhibition of p53 193 , split prime editor with untethered reverse transcriptase 185,194 or split-intein prime editor 187 , removing the ribonuclease H domain of M-MLV RT and incorporation of a viral nucleocapsid protein with nucleic acid chaperone activity 195 . Moreover, Cas9 nuclease-based prime editors have been shown to improve PE efficiency, but it comes at the expense of product purity 188,196,197 .…”