Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

Abstract

Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.

Figure 1.. Initial screening for activity of CODE candidates.

(a) Schematic of the development of CODEs. CODEs consist of a nCas9-DNAP fusion protein and a chimeric pegRNA (cpeg) containing a guide RNA and ssDNA template with intended edits and primer binding site (PBS). (b) Architecture of bacterial expression plasmids of CODEs. The editor expression is driven by T7 promoter, and 6x Histidine tag is located at the C-terminus is employed for purification purposes. (c) Construction of HEK293T reporter cell line supporting base conversion via prime editing or CODE. (d) Schematic of the workflow for nucleofection of CODEs and cpegRNA into HEK293T reporter cell line. (e) Percentage of mCherry activation of CODE candidates and the control engineered PE2 system. Error bars represent ± SD, where n = 3 biological replicates.

Figure 2.. Engineering of T4 and Bst chimeric oligonucleotide-directed editors for improved editing.

(a) Architecture of engineered CODE-T4 editors with domain rearrangement strategies. (b) Percentage of mCherry activation of the CODE-T4 variants in (a). (c) Engineering attempts to alter the T4 DNAP processivity and fidelity to create improved CODE-T4 variants beneficial mutations. (d). (e) Optimization of amino acid linker length between nCas9 and T4 DNAP in the fusion construct. (f) Engineering attempts to alter the Bst-LF DNAP thermostability to create improved CODE-Bst variants with beneficial mutations. (g) Percentage of mCherry activation of the CODE-T4 variants in (f). Error bars represent ± SD, where n = 3 biological replicates.

Figure 3.. In-house synthesis of cpegRNA ligation reaction.

(a) Schematic of the T4 RNA Ligase I-mediated cpegRNA synthesis. (b) Representative of denaturing gel showing successful ligation of sgRNA and ssDNA oligo to generate cpegRNA that targets mCherry gene. (c) Visualization of HEK293T cells by fluorescence microscopy showing the mCherry activation by PE2 and engineered CODE-Bst variants with ligated cpegRNA. Cells were transfected with prime editors and CODEs 72 hours prior to imaging. (d) Quantification of mCherry activation in (c) via flow cytometry. (e) Schematic of the workflow for transfection of plasmid encoding human codon-optimized CODE and synthetic or ligated cpegRNA. (f) and (g) Efficiency of intended and unintended modifications of PE2 and CODE2 at MECP2 and DNMT1 loci, respectively. Error bars represent ± SD, where n = 3 biological replicates.

Figure 4.. Efficient chimeric oligonucleotide-directed editing of endogenous gene loci with CODEMax and CODEMax(exo+).

(a) Architecture of plasmid encoding CODEMax and CODEMax(exo+). (b) Alphafold3 predicted structure of the CODEMax(exo+) in complex with cpegRNA and target dsDNA. (c)-(h) Endogenous targeting of CODEMax and CODEMax(exo+) at various gene loci in comparison with PE2 and PEMax. Error bars represent ± SD, where n = 3 technical replicates.