Ligase-mediated programmable genomic integration (L-PGI)

Abstract

Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.

Fig. 1. Overview of ligase-mediated programmable genomic integration (L-PGI) mechanism, biochemical proof of concept, and optimizations in a HEK293T GFP reporter cell line.

A Schematic diagram illustrating the L-PGI editing complex and binding of all hybridization domains in oligonucleotides with star indicating ligation site. B Diagram of edit incorporation for an A to C transversion. The donor DNA (green) contains the desired edit with homologous sequence arms to the left and the right of the base edit. After ligation, the donor competes with the endogenous strand for genome incorporation via the mismatch repair (MMR) pathway. C Validation of edit strategy using recombinant protein and synthetic oligonucleotides in vitro visualized by SDS-PAGE. Lane 1: 50 nt and 200 nt DNA fragment positive controls representing the expected edit products. Lanes 2–5: L-PGI reactions containing (+) or excluding components (-) as labeled. Lane 3 (red) shows expected edit product only when all components are included. D Optimization of DBS/SBS length with statistical comparison between 24 vs 26 nt and 26 vs 32 nt. E Optimization of FBS length with statistical comparison of 9 vs 11 nt and 11 vs 13 nt. F Effect of splint GBS and lmgRNA SBS lengths. Red = locked nucleic acid (LNA), blue = 2’ O-methylation, purple = ribonucleic acid (RNA), black = DNA base, and * = phosphorothioate bond. Nucleotide sequences are shown as they are bound to each other and in some cases a part of the GBS or SBS functions as a single stranded linker. All splints include the same DBS and FBS (not shown) and lmgRNAs include identical scaffolds and spacers (not shown). G Testing linked and split mRNA architecture with varying placements of leucine zippers (LZ), P2A, or XTEN linkers on nCas9 and either T4, SplintR, or human ligase IV (hLig4) truncations. Untreated control represents untransfected cells. D–G utilize the HEK293T GFP reporter cell line assayed by flow cytometry and show results of biological replicates n = 3. Ordinary one-way ANOVA without matching for Gaussian distributions used to calculate all p values. H Summary of optimization results. All error bars represent standard deviation. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-3, 6.

Fig. 2. Prime editing (PE) vs L-PGI for installation of point mutations in 3 disease-relevant loci in wild-type primary human hepatocytes (PHH).

Results shown for n = 3 biological replicates for all experiments unless otherwise indicated. A Efficiency and fidelity of 2 nt substitution of +5 G to T and +8 G to T to install H1069Q with silent mutation in ATP7B assayed by next generation sequencing (NGS). L-PGI was compared to PE2, PE3, and PEMax. Ordinary one-way ANOVA without matching with Gaussian distribution used to calculate p value for efficiency comparison between PEMax and L-PGI. B Comparison of engineered nCas9-RT variant controls with published mRNA sequences for placement of 38 bp Bxb1 attachment site (attB) in F9 intron 1 via twin PE mechanism using paired guides with 20 overlap in PHH assayed by digital droplet PCR (ddPCR). Unpaired T test and ordinary one-way ANOVA with Gaussian distribution performed, respectively. n = 2 for PE6 test condition only. C Efficiency and fidelity of 2 nt substitution of UGC to UAU to install C282Y with silent mutation in HFE assayed by NGS. L-PGI is compared to PE-TB2 (nCas9 fused to engineered RT). Ordinary one-way ANOVA performed for efficiency to calculate p values, ns indicates not significant. n = 2 for L-PGI test condition only. D Schematic map showing location of guide target window in a 900 bp range spanning the transcription start site (TSS) preceding exon 2 of APOA1. E Efficiency of 1–3 nt substitutions performed by either PE or L-PGI shown side by side using the same spacer sequences for either pegRNA or lmgRNA and identical ngRNA by Sanger Sequencing and inference of CRISPR edits (ICE) analysis against untreated control. Paired T test performed with estimation plot showing up to 54.3 percentage gain in efficiency with average gain of 13.1 across all tested spacers. F Edit quality comparison for subset of APOA1 spacers showing comparable fidelity across loci and methods. SP4275 PE-TB3, SP4286, and SP4275 L-PGI test conditions contain n = 2 biological replicates. All error bars represent standard deviation. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4.

Fig. 3. Comparison of PE vs L-PGI for multi-nt corrections in PHH.

A Illustration showing the 14 nt edit with and without excision of endogenous sequence representing end cases preserving either endogenous sequence or reading frame. The endogenous sequence downstream of the nick is shown in blue and red. The donor contains the 14 nt insertion followed by sequence homologous to the red for sequence replacement or to the blue for sequence insertion. B Highest total efficiencies and maximum fidelities observed using either sequence replacement or insertion observed using either PE-TB2 or L-PGI in PAH intron 1 obtained through component ratio optimization. Ordinary one-way ANOVA with Gaussian distribution performed for biological replicates n = 2 with p values shown. Efficiencies and fidelity vary depending on component ratio, for complete data set refer to Supplementary Fig. 3. C Comparison of lowest indel generation rates observed between PE-TB2 and L-PGI. Indel rate is calculated as the rate of total erroneous deletions and insertions occurring within the nick to nick edit window normalized to total edited reads. P values determined with Ordinary one-way ANOVA with Gaussian distribution for multiple comparisons. n = 3 for HFE 2nt edit PE-TB2 condition and 2 for all other test conditions. D Representative NGS paired-end alignment showing top 15 reads each of 14 nt insertions in PAH intron 1 of PHH using L-PGI (top) and PE-TB2 (bottom). Black bars indicate deletions, highlighted bases show substitutions, and underlined bases contain insertions. In L-PGI the dominant errors are point substitutions in the donor region (24 nt in red in reference alignment) and excisions between the end of the donor and the nicking site on the opposite strand (2 nt in red). PE results in additional insertions and deletions in the edit window. E Comparison of 38 nt Bxb1 attB placement efficiency in PAH intron 1 using L-PGI or PE-TB2 by NGS showing correct editing rate only. Ordinary one-way ANOVA with Gaussian distribution performed with n = 3. All error bars represent standard deviation. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4.

Fig. 4. Paired L-PGI (pL-PGI) design and efficiencies across cell types for Bxb1 and Pa01 attB placement and excision.

A Schematic diagram of paired L-PGI complexes targeting opposite strands to install paired reverse complementary donors. B Design strategies demonstrating replacement type edit (left) by encoding the desired edit in reverse complementary partial overlap donors or deletion type edit (right) by having donors homologous to the genome flanking the sequence to be excised. C Pa01 33 bp attB placement efficiencies using paired complexes PE2, PEMax, or pL-PGI in NOLC1 of HEK293T measured by NGS for biological replicates n = 3. Control defined as untransfected cells. Ordinary one-way ANOVA for multiple comparisons with Gaussian distribution performed for precise edit efficiencies by edit method with p values shown. D Efficiencies of Pa01 33 bp attB placement in NOLC1 of hematopoietic stem cells (HSCs), Bxb1 38 bp attB placement in B2M of induced pluripotent stem cells (iPSCs), and 42 bp edit containing Bxb1 38 bp attB with TAAT stop codon in CIITA of iPSCs, all results assayed by ddPCR. Control conditions received electroporation only with no transfection reagents. Each condition performed once n = 1. E Efficiencies of 175 bp excision at VEGFA of HEK293T, iPSC, and PHH by either dual PE2 or pL-PGI by NGS for biological replicates n = 3. All error bars represent standard deviation. Source data are provided in Source Data file. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4.

Fig. 5. pL-PGI attB placement optimizations across therapeutic loci and implementation of pL-PGI for Bxb1-mediated integration in PHH.

All results show biological replicates of n = 3 unless otherwise stated. A Efficiency and fidelity of attB placement with symmetrically varying overlap for forward and reverse donors in F9. Donor and splint DBS were kept locked to maintain full donor splint hybridization (24, 29, 38 nt for 10, 20, and 38 overlap, respectively). Control is untreated cells. Each condition for efficiency testing performed n = 2 and for fidelity, n = 6. % edit quantified as precise editing only and % fidelity quantified as precise editing out of total editing for all groups. Ordinary one-way ANOVA for multiple comparisons with Gaussian distribution used to calculate p values. B Application of 10 overlap pL-PGI for attB placement in F9, Albumin (ALB), and PAH in PHH versus dual 20 overlap PE-TB2 showing frequency of precise editing only by NGS. C Schematic illustration of 2-step attB placement followed by Bxb1-mediated integration of viral cargo showing expected genomic products. ITR = inverted terminal repeats, attP = attachment site P, attL = post recombination left junction, attR = post recombination right junction. D Residual unconverted attB and total gene integration efficiency of helper-dependent adenovirus (HDAd) comparing attB placed by either dual PE or pL-PGI. Sum of both bars represents total edit efficiency for each attB placement method. Control is untreated cells. E Comparison of attB conversion rate (calculated as ratio of integration to integration + residual attB) between dual PE and pL-PGI across both left and right junctions. Ordinary one-way ANOVA for multiple comparisons used for calculation of p values. All error bars indicate standard deviation. Source data are provided in Source Data file. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4, 7.

Fig. 6. Translation of pL-PGI across primary cell species in vitro, delivery with lipid nanoparticles (LNP), and efficiency in mice.

A Perfect edit and indel efficiencies at PAH intron 1 in PHH, primary cynomolgus hepatocytes (PCH), and primary mouse hepatocytes (PMH), respectively using guides targeting the identical nicking sites for both edit methods in each comparison. PHH and PMH show results of biological replicates n = 3 and PCH results of biological replicates n = 2. Ordinary one-way ANOVA with Gaussian distribution performed for all analyses. B Fidelity comparison between pL-PGI and dual PE-TB2 for the same edits in PHH, PCH, and PMH, respectively, calculated by taking the percent of perfect beacons out of total edits. PHH and PMH show results of biological replicates n = 3 and PCH results of biological replicates n = 2. C pL-PGI LNP formulation payload schematic depicting all in one (AIO) delivery and split delivery. AIO: single particle with all 8 components: nCas9 mRNA (1), ligase mRNA (2), forward lmgRNA (3), reverse lmgRNA (4), forward splint (5), reverse splint (6), forward donor (7), and reverse donor (8). Split: two particles each containing half of both mRNAs and either forward guide, forward splint, forward donor or reverse guide, reverse splint, and reverse donor. D Potency of dual PE-TB2 AIO versus AIO and split pL-PGI LNP targeting F9 in PHH assayed by ddPCR showing biological replicates n = 3 for each treatment dose. 4-parameter nonlinear curve calculated showing EC50 (ng/well) and R squared goodness of fit for each group. E Dual PE-TB3 vs pL-PGI for attB placement in mouse PAH assayed by ddPCR. Ordinary one-way ANOVA performed on biological replicates n = 5 for all groups. Control group treated with saline vehicle. F PE-TB3 vs L-PGI for insertion of a 14 nt edit in mouse PAH assayed by ddPCR showing 2-fold gain in efficiency. Ordinary one-way ANOVA performed on biological replicates n = 5. G Alanine transaminase (ALT) and aspartate transaminase (AST) levels in units/L (U/L) in mouse serum collected 24 h and 7 days after LNP injection. Sampled from individual animals n = 3. Ordinary one-way ANOVA performed for p values. All error bars indicate standard deviation. All one-way ANOVA tests assume Gaussian distribution with no matching. Source data are provided in Source Data file. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4, 7.