PINE-TREE enables highly efficient genetic modification of human cell lines

Abstract

Prime editing technologies enable precise genome editing without the caveats of CRISPR nuclease-based methods. Nonetheless, current approaches to identify and isolate prime-edited cell populations are inefficient. Here, we established a fluorescence-based system, prime-induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), for real-time enrichment of prime-edited cell populations. We demonstrated the broad utility of PINE-TREE for highly efficient introduction of substitutions, insertions, and deletions at various genomic loci. Finally, we employ PINE-TREE to rapidly and efficiently generate clonal isogenic human pluripotent stem cell lines, a cell type recalcitrant to genome editing.

Keywords

• MT: RNA/DNA
• CRISPR
• genome modification
• prime editing
• human pluripotent stem cells