Main

In vivo CRISPR base editing is an emerging therapeutic approach to efficiently and precisely make genomic alterations, including the direct correction of pathogenic variants, in a patient’s body1,2. Early results from a clinical trial of base editing to inactivate the PCSK9 gene in patients with familial hypercholesterolaemia have provided a strong proof of concept of the efficacy of adenine base editing in the human liver in vivo3. These results are motivating the development of liver-centred base-editing therapies to address a wide variety of genetic disorders. In previous studies, we have demonstrated the ability of in vivo base editing to definitively address two such disorders in postnatal or prenatal mouse models: correction of the recurrent PAH P281L variant or PAH R408W variant in humanized mouse models of phenylketonuria (PKU), a metabolic disorder caused by increased blood phenylalanine (Phe) levels resulting from a defective phenylalanine hydroxylase (PAH) enzyme4,5; and inactivation of the mouse ortholog of the HPD gene in a mouse of model of HT1, a metabolic disorder caused by toxic metabolites resulting from a defective fumarylacetoacetate hydroxylase (FAH) enzyme, addressable by blocking the upstream 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme6. Pseudoxanthoma elasticum (PXE) is a connective tissue disorder marked by mineralization in various body tissues, associated with decreased blood pyrophosphate levels resulting from a defective ATP binding cassette subfamily C member 6 (ABCC6) transporter most highly expressed in the liver. The ABCC6 R1164X variant is one of the most recurrent variants identified in patients with PXE7.

The eighth-generation adenine base editor (ABE), ABE8.8-m (ABE8.8), has now been validated in preclinical non-human primate studies and in the aforementioned clinical trial3,8,9. As with other ABEs, it can use its adenosine deaminase domain to efficiently catalyse A→G edits on the sense or antisense strands of genes and correct a substantial proportion of human pathogenic variants. ABE8.8 also has among the narrowest editing windows of the eighth-generation ABEs10. If the position of the target adenine base lies within the window of ABE8.8, a narrow window limits the potential for two undesirable consequences of base editing: bystander editing and off-target editing. Bystander editing of one or more adenine bases near the target adenine base can be counterproductive if it introduces new pathogenic variants alongside the desired corrective editing of the original pathogenic variant. Off-target editing, a general concern with any type of CRISPR editing, entails mutagenesis at genomic sites other than the desired on-target site. Besides the use of a narrow-window ABE, strategies that can simultaneously reduce off-target editing and bystander editing would be of substantial value in improving the safety and efficiency of ABE therapies.

In this Article we evaluate clinical lead guide RNAs (gRNAs) that, in combination with an ABE, achieve therapeutically desirable edits in PAH, ABCC6 or HPD in hepatocytes. To reduce off-target editing by these gRNAs, we explored a previously reported strategy, the in vitro or in cellulo use of hybrid gRNAs in which certain positions of the spacer sequence are substituted with DNA nucleotides11,12,13,14,15,16—a strategy compatible with the use of mRNA-lipid nanoparticles (LNPs) for in vivo delivery. In systematically assessing the effects of a series of hybrid gRNAs on the off-target profiles of clinical lead gRNAs, we unexpectedly observed that hybrid gRNAs have the potential to reduce bystander editing as well. Importantly, we found that hybrid gRNAs that reduced off-target editing by the lead PAH P281L-correcting and ABCC6 R1164X-correcting ABE/gRNA combinations in vivo, in PKU and PXE humanized mouse models, did so without compromising in vivo editing efficiency. On the contrary, the hybrid gRNAs <q>significantly increased the desired corrective editing in the liver while simultaneously reducing unwanted bystander editing.

Results

Optimizing correction of the PAH P281L variant in cellulo

In a previous study we characterized a clinical lead gRNA (hereon PAH1) targeting the human-specific protospacer sequence TCACAGTTCGGGGGTATACA, with the corresponding protospacer-adjacent motif (PAM) TGG, to correct the PAH P281L variant4 (Fig. 1a). The variant adenine base lies in position 5 of the protospacer sequence. A potential bystander adenine base lies nearby in position 3, and bystander A→G editing would result in a splice site disruption that has been reported to be pathogenic for PKU17 and thus would be undesirable. In combination with ABE8.8, the PAH1 gRNA results in efficient corrective editing of the P281L variant in cellulo (in HuH-7 hepatocytes homozygous for the P281L variant, derived from the HuH-7 human hepatoma cell line) and in vivo (in mouse liver), albeit with sizeable levels of bystander editing4.

Fig. 1: Assessment of on-target and off-target editing by candidate clinical lead gRNAs.
figure 1

a, Schematics of the genomic sites of the five loci that are evaluated in this study, adapted from the UCSC Genome Browser (GRCh38/hg38). Each vertical yellow bar indicates either the G altered to A (in red) by a pathogenic variant, or the target A (in red) at a splice site intended for disruption. Each horizontal grey bar indicates the protospacer (thick) and protospacer-adjacent motif (PAM) (thin) sequences targeted by the respective gRNA. b, Schematic of the ONE-seq methodology.

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Most CRISPR-based off-target assay techniques are designed to detect double-strand breaks in DNA, whether in vitro or in cellulo. However, base editors such as ABE8.8 do not directly make double-strand breaks, and so conventional off-target assays such as GUIDE-seq do not accurately reflect off-target editing by base editors. We therefore utilized an ABE-tailored version of OligoNucleotide Enrichment and sequencing (ONE-seq) to nominate off-target sites8,18, followed by genomic sequencing to verify these candidate sites (Fig. 1b) (Supplementary Note 1).

For the ABE8.8/PAH1 combination, we had previously performed a ONE-seq analysis followed by targeted amplicon sequencing of ~50 top-ranked sites that verified one genomic site of low-level off-target mutagenesis in HuH-7 hepatocytes4. We now undertake a deeper analysis of the ONE-seq rank-ordered list, using hybrid capture sequencing to assess the entire set of 280 genomic sites with ONE-seq scores greater than 0.01 (normalized to the on-target site having a score of 1.0) (Supplementary Table 1). We verified an additional six sites of off-target mutagenesis in ABE8.8/PAH1 mRNA/gRNA-transfected P281L HuH-7 hepatocytes (Fig. 2a). None of the seven sites raises a concern of oncogenic risk (Supplementary Note 2). The third verified off-target site (PAH1_OT3) displayed the most off-target editing, with 1.3% editing.

Fig. 2: Optimizing correction of the PAH P281L variant in cellulo and in vivo.
figure 2

a, Hybrid capture sequencing of ONE-seq-nominated sites in treated versus control PAH P281L HuH-7 cells with PAH1 gRNA in combination with ABE8.8 mRNA (n = 3 treated and n = 3 untreated biological replicates), with seven human sites verified to have off-target editing. b, Spacer sequences of PAH1 standard and hybrid gRNAs. rN, ribonucleotide; dN, deoxyribonucleotide; m, 2’-O-methylation; *, phosphorothioate linkage. DNA substitutions are in bold red and underlined. c, A-to-G editing of the PAH on-target site (correction of P281L variant only, or correction plus unwanted nonsynonymous bystander editing) or verified PAH1 human off-target site (PAH1_OT3) in PAH P281L HuH-7 cells treated with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 2–3 biological replicates per condition; exact numbers are provided in the source data). d, Number of sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. e, A-to-G editing of the PAH on-target site or verified PAH1 human off-target site in PAH P281L HuH-7 cells treated with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 11–12 biological replicates per condition; exact numbers are provided in the source data). f, Number of sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. g, Total A-to-G editing of the PAH on-target site or any of seven verified PAH1 human off-target sites in untreated versus treated PAH P281L HuH-7 cells with PAH1 standard or PAH1_hyb22 gRNA in combination with ABE8.8 mRNA (n = 11–12 biological replicates per condition; exact numbers are provided in the source data). h, Number of variant (non-reference-genome) sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. i, A-to-G editing of the PAH on-target site or verified PAH1 mouse off-target site (PAH1_mOT3) in homozygous PAH P281L mice treated with 2.5 mg kg−1 dose of LNPs with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 4–5 animals per group; exact numbers are provided in the source data). In all relevant graphs, means are shown. P values were calculated with a two-sided Mann–Whitney U test.

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To assess whether hybrid gRNAs could reduce off-target editing at the verified sites, we designed a series of 21 synthetic hybrid gRNAs (PAH1_hyb1–PAH1_hyb21) with single, double or triple DNA nucleotide substitutions ranging from positions 3 to 10 in the spacer sequence (Fig. 2b). We transfected ABE8.8 mRNA in combination with the PAH1 standard gRNA and each of the hybrid gRNAs into P281L HuH-7 hepatocytes and assessed for (1) on-target P281L corrective editing, (2) bystander editing at the on-target site and (3) PAH1_OT3 off-target editing in each of the samples (Fig. 2c). Although most of the PAH1 hybrid gRNAs had comparable on-target editing to the PAH1 standard gRNA (~90%), several had modestly decreased editing (as low as ~80%). Notably, and unexpectedly, most of the hybrid gRNAs reduced bystander editing (from 4.4% with the standard gRNA to as low as ~1%). The PAH1 hybrid gRNAs had highly varied effects on PAH1_OT3 off-target editing. Although triple DNA nucleotide substitutions generally reduced PAH1_OT3 off-target editing more than single or double substitutions, a couple of gRNAs with triple substitutions had greater PAH1_OT3 off-target editing than the standard gRNA. None of the 21 PAH1 hybrid gRNAs fully eliminated PAH1_OT3 off-target editing. As an orthogonal method of off-target profiling, we performed ONE-seq with ABE8.8 and each of the 21 PAH1 hybrid gRNAs (Fig. 2d). We used the number of genomic sites with ONE-seq scores greater than 0.01 as a metric of gRNA specificity—the fewer the sites, the less potential for off-target editing. There was good agreement between the results of the site-specific PAH1_OT3 analysis and the more general ONE-seq off-target profiling for PAH1 hybrid gRNAs.

To further reduce off-target editing, we combined triple and double substitutions within a single hybrid gRNA. We chose the triple substitutions of PAH_hyb17 (positions 4, 5 and 6) or PAH_hyb16 (positions 3, 4 and 5), which maximally reduced PAH1_OT3 off-target editing and maximally reduced bystander editing while preserving on-target editing, and added the double substitutions of PAH_hyb15 (positions 9 and 10), which substantially reduced PAH1_OT3 off-target editing and bystander editing, yielding PAH1_hyb22 and PAH1_hyb23. We added an additional substitution in position 11 to PAH1_hyb23, yielding PAH1_hyb24. We compared these three hybrid gRNAs directly against the PAH1 standard gRNA and negative controls via mRNA/gRNA transfection in P281L HuH-7 hepatocytes, and all three significantly reduced PAH1_OT3 off-target editing and bystander editing (Fig. 2e). ONE-seq confirmed the improved off-target profiles of the three new hybrid gRNAs relative to the 21 original hybrid gRNAs; indeed, PAH1_hyb24 had zero off-target sites with ONE-seq scores greater than 0.01 (Fig. 2f). At all seven verified PAH1 off-target sites, OT1–OT7, PAH1_hyb22 reduced the detectable off-target editing to the background levels of the negative controls (Fig. 2g).

All standard off-target assessment techniques share a critical limitation: each is tied to the specific individual genome represented by the cells or by the in vitro genomic DNA sample used for analysis. For this reason, most off-target analyses have simply ignored the potential for naturally occurring human genetic variation to create novel off-target editing sites in some patients. A cautionary tale is provided by the finding that the gRNA used in the recently approved CRISPR-based therapy for sickle cell disease, exa-cel, has substantial off-target editing in hematopoietic stem cells at a genomic site created by a genetic variant present in ~10% of individuals of African ancestry, resulting in both indel mutations and chromosomal rearrangements19. The ONE-seq technique is uniquely capable of performing a variant-aware off-target analysis, accommodating naturally occurring human genetic variation by using oligonucleotide libraries designed not just using the reference human genome but also incorporating data from the 1000 Genomes Project, the Human Genome Diversity Project, and so on. We used the bioinformatic tool CRISPRme19 to design a variant-aware ONE-seq library for the PAH1 protospacer with ~7,000 non-reference-genome oligonucleotides. In performing a variant-aware ONE-seq experiment with the ABE8.8/PAH1 combination, we identified 40 variant sites with ONE-seq scores greater than 0.01 (Fig. 2h). One of the top variant sites, with a ONE-seq score of 0.19, is a rare singleton 1-bp deletion that alters a reference genomic site that has seven mismatches with the on-target protospacer, with an NTG protospacer-adjacent motif (PAM), into a site that has three mismatches, with an NGG PAM (Extended Data Fig. 1). Another top variant site, with a ONE-seq score of 0.11, is common enough to have been previously catalogued as rs76813758, a single-nucleotide variant that alters a reference genomic site that has four mismatches with the on-target protospacer, with an NGG PAM, into a site that has three mismatches, with an NGG PAM. This variant is present in ~8% of individuals of African ancestry and ~3% of individuals of European ancestry (Extended Data Fig. 1).

One limitation of variant-aware off-target analysis is that, upon identifying candidate variant sites, it can be challenging to verify whether editing actually occurs at any of those sites in the therapeutically relevant cells (for example, hepatocytes) if there is no way to obtain such cells from individuals with those variants. We reasoned that the use of hybrid gRNAs should reduce the potential for off-target editing not only at reference genomic sites, but also at variant sites, mitigating the need to evaluate variant sites in cellulo. Accordingly, we performed variant-aware ONE-seq experiments for the ABE8.8/PAH1_hyb22 and ABE8.8/PAH1_hyb24 combinations (Fig. 2h and Extended Data Fig. 1). With PAH1_hyb22, there were just three variant sites with ONE-seq scores greater than 0.01, and with PAH1_hyb24, there were no variant sites with ONE-seq scores greater than 0.01. For rs76813758, the ONE-seq score dropped from 0.11 to 0.004 with PAH1_hyb22 and to 0.0001 with PAH1_hyb24 (1/10,000th of the in vitro editing activity of the on-target site, within the background of the assay).

Optimizing correction of the PAH P281L variant in vivo

Although our rational exploration of hybrid gRNAs was successful in rendering editing at the seven verified PAH1 off-target sites undetectable in cellulo, while preserving on-target editing efficiency in cellulo, it remained to be answered whether hybrid gRNAs could function as effectively in vivo. We formulated LNPs with ABE8.8 mRNA and PAH1 standard gRNA, PAH1_hyb22 gRNA, PAH1_hyb23 or PAH1_hyb24, exactly paralleling LNPs we used in a recently published study4. We administered the LNPs to homozygous humanized P281L PKU mice at a dose of 2.5 mg kg−1. We observed that only the PAH1_hyb23 and PAH1_hyb24 gRNAs resulted in complete normalization of blood Phe levels (mean <125 µmol l−1) by 48 h after treatment, significantly outperforming the PAH1 standard gRNA (Extended Data Fig. 2). With ABE8.8/PAH1 LNPs, there was 40% (mean) whole-liver P281L corrective editing, and with each of the hybrid gRNAs, there was mean 50–60% editing, establishing that the use of the hybrid gRNAs did not compromise, and in fact significantly improved, on-target editing (Fig. 2i). The increased on-target editing was accompanied by significantly reduced bystander editing (mean 0.8% editing with standard gRNA compared to mean 0.2–0.3% editing with hybrid gRNAs).

We performed ONE-seq for the ABE8.8/PAH1 combination against the reference mouse genome (Supplementary Table 2). In interrogating the candidate sites with the top ONE-seq scores in liver samples from the LNP-treated mice, we verified a site (PAH1_mOT3) with mean 2.1% off-target editing with the PAH1 standard gRNA (Fig. 2i). PAH1_hyb22 gRNA, PAH1_hyb23 and PAH1_hyb24 all significantly reduced the off-target editing at this site (<0.2%).

Optimizing correction of the ABCC6 R1164X variant in cellulo

Of the recurrent ABCC6 variants causative of PXE, we focused on the R1164X variant because of features suggesting it would be amenable to adenine base editing (Figs. 1a and 3a), namely the positioning vis-à-vis a TGG PAM compatible with a gRNA (designated PXE1) with protospacer sequence GTCACGGGAAACTGATCCTC, the variant adenine base lying in position 4, and potential bystander adenine bases lying in positions 9, 10 and 11, outside the reported editing window of ABE8.8. We used prime editing to generate a homozygous R1164X HuH-7 cell line (Supplementary Note 3). Transfection of the cell line with ABE8.8 mRNA and the PXE1 gRNA achieved substantial corrective editing of the R1164X variant (32%), albeit with a moderate amount of unwanted bystander editing (6.3%) (Fig. 3b). For the ABE8.8/PXE1 combination, we performed a ONE-seq analysis followed by targeted amplicon sequencing of top-ranked sites that verified six genomic sites of off-target mutagenesis in HuH-7 cells (Supplementary Table 3). None of the six sites raises a concern of oncogenic risk (Supplementary Note 2). The first and second verified off-target sites (PXE1_OT1 and PXE1_OT2) displayed the most off-target editing.

Fig. 3: Optimizing correction of the ABCC6 R1164X variant in cellulo and in vivo.
figure 3

a, Spacer sequences of PXE1 standard and hybrid gRNAs. rN, ribonucleotide; dN, deoxyribonucleotide; m, 2’-O-methylation; *, phosphorothioate linkage. DNA substitutions are in bold red and underlined. b, A-to-G editing of the ABCC6 on-target site (correction of the R1164X variant only, or correction plus unwanted nonsynonymous bystander editing) or either of two verified PXE1 human off-target sites (PXE1_OT1, PXE_OT2) in ABCC6 R1164X HuH-7 cells treated with PXE1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 2–3 biological replicates per condition; exact numbers are provided in the source data). c, Number of sites with a ONE-seq score of >0.01 for each indicated PXE1 standard or hybrid gRNA with ABE8.8 protein. d, A-to-G editing of the ABCC6 on-target site or either of two verified PXE1 human off-target sites in ABCC6 R1164X HuH-7 cells treated with PXE1 standard or PXE1_hyb18 gRNA in combination with ABE8.8 mRNA (n = 5–6 biological replicates per condition; exact numbers are provided in the source data). e, A-to-G editing of the ABCC6 on-target site or verified PXE1 mouse off-target site (PXE1_mOT24) in homozygous ABCC6 R1164X mice treated with LNPs (2.5 mg kg−1 dose) with PXE1 standard or PXE1_hyb18 gRNA in combination with ABE8.8 mRNA (n = 6 animals per group). In all relevant graphs, means are shown; P values were calculated with a two-sided Mann–Whitney U test.

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As we had done with the PAH1 gRNA, we designed a series of 21 hybrid gRNAs for PXE1 (PXE1_hyb1–PXE1_hyb21) with single, double or triple DNA nucleotide substitutions ranging from positions 3 to 10 in the spacer sequence (Fig. 3a), matching the designs of the PAH1 hybrid gRNAs. Following transfection of ABE8.8 mRNA and each of the gRNAs into R1164X HuH-7 hepatocytes, we found that all of the hybrid gRNAs reduced off-target editing at the OT1 and OT2 sites, some of the hybrid gRNAs increased on-target editing (to as high as ~50%), and some of the hybrid gRNAs reduced bystander editing (to as low as ~3%) (Fig. 3b). We performed ONE-seq for each of the hybrid gRNAs and, judging by the number of genomic sites with ONE-seq scores greater than 0.01, improvement of off-target editing was concordant with the effects on the individual OT1 and OT2 sites (Fig. 3c). We further evaluated PXE1_hyb18 and found that it significantly increased on-target editing and significantly, though not entirely, reduced off-target editing at the OT1 and OT2 sites (Fig. 3d). It is possible that additional DNA nucleotide substitutions would yield hybrid gRNAs that further reduce off-target editing.

Optimizing correction of the ABCC6 R1164X variant in vivo

We used CRISPR–Cas9 targeting in mouse embryos to generate a humanized PXE model, in the C57BL/6J background, in which we replaced a small portion of the endogenous mouse Abcc6 exon 24 with the orthologous human sequence spanning the PXE1 protospacer/PAM sequences and containing the R1164X variant (Supplementary Note 4). Consistent with previous studies of Abcc6 knockout mice7, homozygous R1164X mice had reduced blood pyrophosphate levels compared to wild-type littermates (Extended Data Fig. 3).

We formulated LNPs with ABE8.8 mRNA and either PXE1 standard gRNA or PXE1_hyb18 gRNA. We administered the LNPs to homozygous R1164X mice at a dose of 2.5 mg kg−1. With ABE8.8/PXE1 LNPs, there was mean 24% whole-liver P281L corrective editing in the absence of bystander editing, and mean 3.5% corrective editing with bystander editing; with the hybrid gRNA, there was 29% and 2.1% editing, respectively (Fig. 3e). The PXE1 hybrid gRNA significantly increased the on-target editing and reduced bystander editing in vivo, as was observed with PAH1 hybrid gRNAs in vivo. LNP treatment also largely normalized the blood pyrophosphate levels in the homozygous R1164X mice (Extended Data Fig. 3a).

We performed ONE-seq for the ABE8.8/PXE1 combination against the reference mouse genome (Supplementary Table 4). In interrogating the candidate sites with the top ONE-seq scores in liver samples from the LNP-treated mice, we verified a site (PXE1_mOT24) with mean 8.4% off-target editing with the PXE1 standard gRNA (Fig. 3e). The PXE1_hyb18 gRNA significantly reduced off-target editing at this site, with mean 0.6% editing. We also assessed blood alanine aminotransferase (ALT) and cytokine/chemokine levels in LNP-treated mice, and we observed similar safety profiles with the standard and hybrid gRNAs (Extended Data Fig. 3b and Supplementary Table 5).

Optimizing targeting of two distinct sites in the HPD gene with hybrid gRNAs

Lacking any previous data regarding adenine base editing to inactivate the HPD gene (our previous study assessed only cytosine base editing), we screened for precise disruption of a splice donor or acceptor site in the human HPD gene by adenine base editing (Supplementary Note 5). We observed the highest levels of editing with the gRNAs designated ‘HPD20’, ‘HPD3’, ‘HPD4’ and ‘HPD5’, targeting either the splice acceptor of exon 7 or the splice donor of exon 8 (Fig. 1a and Extended Data Fig. 4a). To evaluate for off-target editing, we used two ONE-seq libraries against the reference human genome: a combined library for the cluster of closely spaced HPD3, HPD4 and HPD5 protospacers (consecutive protospacers shifted by one nucleotide), and a library for the HPD20 protospacer. Targeted amplicon sequencing of top-ranked sites in ABE8.8/gRNA-treated wild-type HuH-7 hepatocytes verified one genomic site of off-target mutagenesis each for HPD3 and HPD4, and none for HPD5 and HPD20 (Extended Data Fig. 4b and Supplementary Tables 6–9).

Because of its more favourable on-target and off-target editing profiles, we prioritized HPD20 for further study as a clinical lead gRNA. We assessed a full series of HPD20 hybrid gRNAs (HPD20_hyb1–HPD20_hyb21) (Fig. 4a), matching the designs of the PAH1 hybrid gRNAs, via ABE8.8 mRNA/gRNA transfection in wild-type HuH-7 hepatocytes, for both editing of the target splice site adenine (in position 6 of the protospacer sequence, TCTGCAGAAAGCACGGGAAC, with a GGG PAM) and bystander editing of positions 8, 9 and/or 10 (Fig. 4b). Because we had not verified any individual genomic sites of off-target editing with the HPD20 standard gRNA, we used ONE-seq to assess the off-target editing profiles of the HPD20 hybrid gRNAs, with many showing decreased off-target potential (that is, number of sites with ONE-seq scores greater than 0.01), but a few showing increased off-target potential (Fig. 4c). Two hybrid gRNAs, HPD20_hyb22 and HPD20_hyb23, with five DNA nucleotide substitutions each, maintained on-target editing, substantially reduced bystander editing, and had the fewest sites with ONE-seq scores greater than 0.01 (Fig. 4a–c).

Fig. 4: Optimizing targeting of two distinct sites in the HPD gene with hybrid gRNAs.
figure 4

a, Spacer sequences of HPD20 standard and hybrid gRNAs. rN, ribonucleotide; dN, deoxyribonucleotide; m, 2’-O-methylation; *, phosphorothioate linkage. DNA substitutions are in bold red and underlined. b, A-to-G editing of HPD on-target site (target adenine only, or target adenine plus bystander editing) in wild-type HuH-7 cells treated with HPD20 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 3 biological replicates per condition). c, Number of sites with a ONE-seq score of >0.01 for each indicated HPD20 standard or hybrid gRNA with ABE8.8 protein. d, Spacer sequences of HPD4 standard and hybrid gRNAs. e, A-to-G editing of the HPD on-target site (target adenine only, or target adenine plus bystander editing) or verified HPD4 off-target site (HPD4_OT13) in wild-type HuH-7 cells treated with HPD4 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 3 biological replicates per condition). In all relevant graphs, means are shown.

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We also assessed the HPD4 gRNA with protospacer sequence CACTCACAGTTTAGGAAGTA and a GGG PAM, with the target adenine base in position 6 and a bystander adenine base in position 8. Having verified a site of off-target editing with the HPD4 standard gRNA (HPD4_OT13) with mean 4.9% editing, we assessed a full series of HPD4 hybrid gRNAs (HPD4_hyb1–HPD4_hyb21) (Fig. 4d), matching the designs of the PAH1 hybrid gRNAs, via ABE8.8 mRNA/gRNA transfection in wild-type HuH-7 hepatocytes (Fig. 4e). All the hybrid gRNAs reduced OT13 off-target editing, some close to the background level, and many hybrid gRNAs reduced bystander editing while maintaining on-target editing.

Optimizing correction of the PAH R408W variant with a PAM-relaxed ABE

In a previous study, we characterized a gRNA (herein termed ‘PAH4’) that corrects the PAH R408W variant, the most frequent variant in PKU, in cellulo and in vivo5. Because there are no NGG PAMs situated appropriately for adenine base editing of the PAH R408W variant, we screened PAM-altered and PAM-relaxed versions20 of ABEs and identified an optimal combination of SpRY-ABE8.8 and protospacer sequence GGCCAAGGTATTGTGGCAGC with an AAA PAM (Fig. 1a). The variant adenine base lies in position 5 of the protospacer sequence. Potential bystander adenine bases lie in positions 6 and 10; bystander editing at position 6 would result in a benign synonymous variant, whereas editing at position 10 would result in a nonsynonymous variant that has been reported to be a pathogenic variant for PKU21,22.

Editors based on the SpRY variant of Streptococcus pyogenes Cas9 are near-PAMless in their ability to engage genomic sites20, greatly expanding their targeting range, but also incurring an increased potential for off-target editing. To evaluate the SpRY-ABE8.8/PAH4 combination, which corrects the PAH R408W variant, we performed ONE-seq against the reference human genome. Rather than interrogate candidate off-target sites nominated by ONE-seq with the PAH4 standard gRNA, we immediately tested a limited series of hybrid gRNAs (double/triple substitutions, PAH4_hyb9–PAH4_hyb21, matching the designs of the analogous PAH1 hybrid gRNAs) with the goal of reducing the potential of SpRY-ABE8.8 for off-target editing, and prospectively winnowing the list of candidate off-target sites for verification (Fig. 5a). Several of the PAH4 hybrid gRNAs displayed increased on-target editing and/or decreased bystander editing at protospacer position 10 (nonsynonymous variant) (Fig. 5b). ONE-seq demonstrated reduced off-target potential with some of these favourable hybrid gRNAs, despite the use of a PAM-relaxed ABE (Fig. 5c).

Fig. 5: Optimizing correction of the PAH R408W variant with a PAM-relaxed ABE.
figure 5

a, Spacer sequences of PAH4 standard and hybrid gRNAs. rN, ribonucleotide; dN, deoxyribonucleotide; m, 2’-O-methylation; *, phosphorothioate linkage. DNA substitutions are in bold red and underlined. b, A-to-G editing of the PAH on-target site (correction of the R408W variant with or without synonymous bystander editing, or correction plus unwanted nonsynonymous bystander editing) in PAH R408W HuH-7 cells treated with PAH4 standard or hybrid gRNA in combination with SpRY-ABE8.8 mRNA (n = 3 biological replicates per condition). Means are shown. c, Number of sites with a ONE-seq score of >0.01 for each indicated PAH4 standard or hybrid gRNA with SpRY-ABE8.8 protein.

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Generalization to additional loci

In comparing our results across the five studied loci, our findings suggest that there will not be a single set of hybrid gRNA modifications that will fully optimize off-target and on-target editing across all genomic loci, although suggestive patterns are evident. The ‘hyb16’ and ‘hyb17’ designs (DNA nucleotide substitutions in protospacer positions 4, 5 and 6, or in positions 5, 6 and 7) substantially reduced off-target editing potential while maintaining on-target editing across all loci, so these designs could be a reasonable starting point for future hybrid gRNA screening campaigns. To test this proposition, we compared standard, hyb16 and hyb17 gRNAs for corrective editing of variants at four additional loci (Extended Data Fig. 5). The CPS1 Q335X variant, the focus of our recent report of a personalized N-of-1 base-editing therapy (‘k-abe’) administered to an infant with carbamoyl phosphate synthetase (CPS1) deficiency23, can be corrected with NGC-ABE8e-V106W (also termed SpCas9-LWKYQS-ABE8e-V106W24) and ‘CPS1-1’ gRNA. The CPS1 R780H variant, another ultra-rare cause of CPS1 deficiency, can be corrected with NGC-ABE8e-V106W and ‘CPS1-2’ gRNA. The ABCC6 R1141X variant is the most frequent variant in PXE and can be corrected with SpRY-ABE8.8 and ‘PXE2’ gRNA. The PAH c.1066–11G>A variant is the second most frequent variant in PKU and can be corrected with SpRY-ABE8.8 and ‘PAH5’ gRNA. In all four cases, one or both hybrid gRNAs maintained or improved the desired corrective editing and reduced bystander editing in cellulo, while substantially improving ONE-seq off-target profiles (Extended Data Fig. 5). We note that the six-month development time of the ‘k-abe’ therapy did not allow for extensive screening of candidate hybrid gRNAs, but in future time-limited N-of-1 development efforts, quick testing and validation of the hyb16 design or hyb17 design would permit it to be incorporated into a therapy administered to a patient.

Discussion

In this study, we have investigated the off-target editing profiles of clinical lead gRNAs in combination with ABEs for the correction of pathogenic variants in PKU and PXE and for inactivation of the HPD gene, a modifier for HT1. We assessed the ability of hybrid gRNAs to mitigate the potential for off-target editing by standard versions of the gRNAs, and found that some hybrid gRNAs can dramatically reduce off-target editing while also reducing bystander editing and maintaining or even enhancing on-target editing efficiency in cellulo and in vivo. Our findings suggest that the use of hybrid gRNAs to reduce off-target and bystander editing by ABEs is generalizable across different target loci, underscoring its potential as a broadly applicable strategy to improve the safety and efficiency of base-editing therapies for various genetic disorders.

We note the limitations of this study. We present data for nine distinct loci in cellulo and two of the loci in vivo, and in each case we were able to identify hybrid gRNAs that had favourable effects on editing specificity and efficiency, but it is possible that hybrid gRNAs would not be beneficial at all loci. All the work in this study focused on adenine base editing. It is possible that the advantages we observed with the use of hybrid gRNAs in cellulo and in vivo are relevant to other editing modalities that rely on the use of gRNAs, such as nuclease editing, prime editing and epigenome editing, but this would require validation for each modality. Although we have demonstrated that the use of a hybrid gRNA can substantially reduce gRNA-dependent off-target editing by an ABE both in cellulo and in vivo, it is not clear that use of a hybrid gRNA would affect gRNA-independent off-target editing, that is, spurious RNA or DNA deamination by the ABE’s adenosine deaminase domain25,26. Further experimentation would be needed to evaluate this possibility. These issues notwithstanding, it is clear that hybrid gRNAs can substantially improve some mRNA-LNP adenine base-editing therapies, such as the ABE8.8/PAH1_hyb24 combination for the treatment of PKU, which we intend to take forward into an early-phase clinical trial in the near future.

Methods

The research described here complied with all relevant regulations. All recombinant DNA research was approved by the Institutional Biosafety Committee at the University of Pennsylvania, where the studies were performed, and were consistent with local, state and federal regulations as applicable, including the National Institutes of Health Guidelines for Research Involving Recombinant of Synthetic Nucleic Acid Molecules. All procedures used in animal studies were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania (protocol #805887), where the studies were performed, and were consistent with local, state and federal regulations as applicable, including the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

RNA production

100-mer gRNAs were chemically synthesized under solid-phase synthesis conditions by a commercial supplier (Agilent or Integrated DNA Technologies) with end-modifications as well as heavy 2’-O-methylribosugar modification as previously described27: for cellular studies, 5’-mX*mX*mX*XXXXXXXXXXXXXXXXXGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*U*U*U-3’; for mouse studies, 5’-mX*mX*mX*XXXXXXXXXXXXXXXXXGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU-3’; where the Xs indicate the spacer sequence positions (specified in Figs. 2b, 3a, 4a,d and 5a), and ‘m’ and * respectively indicate 2’-O-methylation and phosphorothioate linkage. ABE8.8 mRNA, SpRY-ABE8.8 mRNA and NGC-ABE8e-V106W mRNA were produced via in vitro transcription and purification as previously described4,5,23. A plasmid DNA template containing a codon-optimized ABE8.8, SpRY-ABE8.8 or NGC-ABE8e-V106W coding sequence and a 3’-polyadenylate sequence was linearized. An in vitro transcription reaction containing linearized DNA template, T7 RNA polymerase, nucleoside triphosphates (NTPs) and cap analogue was performed to produce mRNA containing N1-methylpseudouridine. After digestion of the DNA template with DNase I, the mRNA product underwent purification and buffer exchange, and the purity of the final mRNA product was assessed with spectrophotometry and capillary gel electrophoresis. Elimination of double-stranded RNA contaminants was assessed using dot blots and transfection into human dendritic cells. Endotoxin content was measured using a chromogenic Limulus amebocyte lysate (LAL) assay. All assays were negative.

LNP formulation

LNPs were formulated as previously described4,5,28, with the lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and PEG-2000 at molar ratios of 50:10:38.5:1.5) being rapidly mixed with an aqueous buffer solution containing ABE8.8 mRNA and a gRNA in a 1:1 ratio by weight in 25 mM sodium acetate (pH 4.0), with an N:P ratio of 5.6. The resulting LNP formulations were subsequently dialysed against sucrose-containing buffer, concentrated using Amicon Ultra-15 mL centrifugal filter units (Millipore Sigma), sterile-filtered using 0.2-µm filters, and frozen until use. The LNPs underwent quality control for particle size (Z-Ave, hydrodynamic diameter), polydispersity index (as determined by dynamic light scattering; Malvern NanoZS Zetasizer) and total RNA encapsulation, as measured by the Quant-iT Ribogreen Assay (Thermo Fisher Scientific).

Culture and transfection of HuH-7 cells

Wild-type HuH-7 cells, PAH P281L homozygous HuH-7 cells4, PAH R408W homozygous HuH-7 cells5, ABCC6 R1164X homozygous HuH-7 cells (Supplementary Note 3) and lentivirus-transduced HuH-7 cells with other variants23 were used for this study. The HuH-7 cells (JCRB0403, Fujifilm) were maintained in Dulbecco’s modified Eagle’s medium (containing 4 mM L-glutamine and 1 g l−1 glucose) with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. The HuH-7 cells were seeded on 24-well plates (Corning) at 1.2 × 105 cells per well. For plasmid transfections, at 3–4 h after seeding, the cells were transfected at approximately 80–90% confluency with 1.5 μl of TransIT-LT1 transfection reagent (MIR2300, Mirus), 0.25 μg of base editor plasmid and 0.25 μg of gRNA plasmid per well, according to the manufacturer’s instructions. An ABE8.8 (ABE8.8-m) expression plasmid10 was used as previously described4, and HPD3, HPD4, HPD5, HPD20 and PXE1 gRNA expression plasmids were newly generated for this study. For mRNA/gRNA transfections, in one tube, 3 µl of Lipofectamine MessengerMax (LMRNA008, Thermo Fisher Scientific) was added to 50 µl of Opti-MEM and incubated for 10 min; in another tube, 0.5 µg of mRNA and 0.5 µg of gRNA was suspended in a total of 50 µl of Opti-MEM. The diluted RNA was added to the tube of diluted MessengerMAX, and the subsequent RNA/MessengerMax/Opti-MEM mixture was incubated for 5 min at room temperature and then added dropwise onto the cells. For each type of transfection, cells were cultured for 72 h after transfection, and then media were removed, cells were washed with 1× DPBS (Corning), and genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions.

ONE-seq

ONE-seq was performed as previously described4,8,18. The human ONE-seq libraries for the PAH1, PAH4, PAH5, PXE1, PXE2, HPD3/HPD4/HPD5 (combined library), HPD20, CPS1-1 and CPS1-2 gRNAs were designed using the GRCh38 Ensembl v98 reference genome (ftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.{1-22,X,Y,MT}.fa,), and the mouse ONE-seq libraries for the PAH1 and PXE1 gRNAs were designed using the GRCm39 Ensembl reference genome (ftp://ftp.ensembl.org/pub/release-113/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.chromosome.{1-19,X,Y,MT}.fa, ftp://ftp.ensembl.org/pub/release-113/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.nonchromosomal.fa). Sites with up to six mismatches and sites with up to four mismatches plus up to two DNA or RNA bulges, compared to the on-target site, were identified with Cas-Designer v1.229. The variant-aware ONE-seq library for the PAH1 gRNA was designed using CRISPRme19, with similar mismatch criteria. The final oligonucleotide sequences were generated with a script18, and the oligonucleotide libraries were synthesized by Twist Biosciences. Recombinant ABE8.8 protein, SpRY-ABE8.8 protein and NGC-ABE8e-V106W protein were produced by GenScript. Duplicate ONE-seq experiments were previously performed for ABE8.8/PAH14; mean ONE-seq scores were used for this study. All other ONE-seq experiments were performed specifically for this study as follows. Each library was PCR-amplified and subjected to 1.25× AMPure XP bead purification. After incubation at 25 °C for 10 min in CutSmart buffer, ribonucleoprotein (RNP) comprising 769 nM recombinant ABE protein and 1.54 µM gRNA was mixed with 100 ng of the purified library and incubated at 37 °C for 8 h. Proteinase K was added to quench the reaction at 37 °C for 45 min, followed by 2× AMPure XP bead purification. The reaction was then serially incubated with EndoV at 37 °C for 30 min, Klenow Fragment (New England Biolabs) at 37 °C for 30 min, and NEBNext Ultra II End Prep Enzyme Mix (New England Biolabs) at 20 °C for 30 min followed by 65 °C for 30 min, with 2× AMPure XP bead purification after each incubation. The reaction was ligated with an annealed adaptor oligonucleotide duplex at 20 °C for 1 h to facilitate PCR amplification of the cleaved library products, followed by 2× AMPure XP bead purification. Size selection of the ligated reaction was performed on a BluePippin system (Sage Science) to isolate DNA of 150–200 bp on a 3% agarose gel cassette, followed by two rounds of PCR amplification to generate a barcoded library, which underwent paired-end sequencing on an Illumina MiSeq System as described below. The analysis pipeline18 used for processing the data assigned a score quantifying the editing efficiency with respect to the on-target site to each potential off-target site. Sites were ranked based on this ONE-seq score, which was used for site prioritization.

Next-generation sequencing

Next-generation sequencing (NGS) was performed as previously described4. For targeted amplicon sequencing, PCR reactions were performed using NEBNext Polymerase (NEB) using the primer sets listed in Supplementary Table 10, designed with Primer3 v4.1.0 (https://primer3.ut.ee/). The following program was used for all genomic DNA PCRs: 98 °C for 20 s, 35× (98 °C for 20 s, 57 °C for 30 s, 72 °C for 10 s), 72 °C for 2 min. PCR products were visualized via capillary electrophoresis (QIAxcel, Qiagen) and then purified and normalized via an NGS Normalization 96-Well Kit (Norgen Biotek Corporation). A secondary barcoding PCR was conducted to add Illumina barcodes (Nextera XT Index Kit V2 Set A and/or Nextera XT Index Kit V2 Set D), using ~15 ng of first-round PCR product as the template, followed by purification and normalization. Final pooled libraries were quantified using a Qubit 3.0 fluorometer (Thermo Fisher Scientific) and then, after denaturation, dilution to 10 pM and supplementation with 15% PhiX, they underwent single-end or paired-end sequencing on an Illumina MiSeq System. The amplicon sequencing data were analysed with CRISPResso2 v230 and scripts to quantify editing. For on-target editing, A-to-G editing was quantified at the site of the target adenine and at the site(s) of each potential bystander adenine in the editing window; for candidate off-target sites, A-to-G editing was quantified throughout the editing window (positions 1–10 of the protospacer sequence). In some cases, PCR amplicons were subjected to confirmatory Sanger sequencing, performed by GENEWIZ.

Hybrid capture sequencing

The PAH1 probe library was generated to cover the 280 ONE-seq-nominated genomic sites with ONE-seq scores greater than 0.01, as well as additional sites. The probe library was generated serially using Agilent SureDesign, where probes were created using three rounds of generation settings. In the initial round, probes were generated with 2× tiling density, moderately stringent masking, and optimized performance with 90 min of hybridization boosting. Regions that were not covered with the initial settings had probes generated with 2× tiling density, least stringent masking and optimized performance with 90 min of hybridization boosting. The remaining uncovered regions had probes generated with 1× tiling density, no masking and no boosting. The hybrid capture procedure was conducted using the SureSelect XT HS2 DNA Reagent Kit (Agilent) as per the manufacturer’s instructions. Briefly, 200 ng of genomic DNA from ABE8.8/PAH1 mRNA/gRNA-treated or control untreated HuH-7 cells, quantified on the TapeStation Genomic DNA ScreenTape platform (Agilent), was prepared and enzymatically fragmented with modifications for 2 × 150 reads. The DNA fragments were then processed for library construction using molecular barcodes. Adaptors were ligated to the DNA fragments, followed by repair, dA-tailing and purification using AMPure XP beads. The libraries were amplified (eight cycles), indexed, and purified with AMPure XP beads, then their quality was assessed via the TapeStation D1000 ScreenTape platform (Agilent); 1,000 ng of each library was prepared and hybridized to the PAH1 probe library, captured using streptavidin beads, amplified (16 cycles), and purified using AMPure XP beads. The final libraries were purified and quantified via the TapeStation High Sensitivity D1000 ScreenTape platform (Agilent). The libraries were pooled and sequenced on an Illumina NovaSeq, performed by Novogene. The resulting reads were processed and analysed using the Agilent Alissa Reporter software with the following settings: normal/tumour pair analysis mode for treated samples, and tumour only for control samples, molecular barcode deduplication mode, duplex MBC deduplication consensus mode, two read pairs per MBC minimum, ten reads minimum coverage depth, three reads minimum supporting variant allele, and 0.001 minimum variant allele frequency. For each of the 280 sites, A-to-G editing was quantified throughout the editing window (Supplementary Table 1). For sites with any position displaying a mean net A-to-G editing level of ≥0.1% in treated versus control samples, the sites were confirmed with targeted amplicon sequencing, as described above. Only 2 out of the 280 sites failed to be captured and sequenced, with both sites being on chromosome Y, which is absent from HuH-7 cells.

Mouse studies

Mouse studies were performed as previously described4. Mice were maintained on a 12-h light/12-h dark cycle, with a temperature range of 65 °F to 75 °F and a humidity range of 40% to 60%, and were fed ad libitum with a chow diet (LabDiet, Laboratory Autoclavable Rodent Diet 5010). Humanized PKU mice homozygous for the PAH P281L allele4 or for the ABCC6 R1164X allele (Supplementary Note 4) on the C57BL/6J background were generated as littermates/colonymates with wild-type C57BL/6J mice. Genotyping was performed using PCR amplification from genomic DNA samples (prepared from clipped tails/ears) followed by Sanger sequencing or NGS. Roughly equal numbers of female and male littermates/colonymates were used for experiments, at ages ranging from six to eight weeks, with random assignment of animals to various experimental groups when applicable, and with collection and analysis of data performed in a blinded fashion when possible. LNPs were administered to mice at a dose of 2.5 mg kg−1 via retro-orbital injection under anaesthesia with 1–2% inhaled isoflurane. Blood samples were collected via the tail tip at various timepoints: pre-treatment or 4 h, 24 h, 48 h or 7 days after treatment. Mice were euthanized one to two weeks after treatment, and eight liver samples (two from each lobe) were obtained on necropsy and processed with the DNeasy Blood and Tissue Kit (QIAGEN) as per the manufacturer’s instructions to isolate genomic DNA. Euthanasia in all instances was achieved via terminal inhalation of carbon dioxide followed by secondary euthanasia through cervical dislocation or decapitation, consistent with the 2020 American Veterinary Medical Association Guidelines on Euthanasia. NGS results from the liver samples were averaged to provide quantification of whole-liver editing.

Measurement of blood phenylalanine

Blood phenylalanine levels were measured by an enzymatic method using the Phenylalanine Assay Kit (MAK005, Millipore Sigma) according to the manufacturers’ instructions. Briefly, plasma samples were collected at timepoints in the early afternoon, to account for diurnal variation in blood phenylalanine levels, and deproteinized with a 10-kDa molecular-weight-cutoff spin filter (CLS431478-25EA, Millipore Sigma) and pretreated with 5 µl of tyrosinase for 10 min at room temperature, before the start of the assay. Reaction mixes were made according to the manufacturers’ instructions, and the fluorescence intensity of each sample was measured (λex = 535/λem = 587 nm).

Measurement of blood pyrophosphate

Plasma samples collected pre-treatment and seven days post-treatment were diluted 1:1 in Tris-acetate pH 8.0 and centrifuged with a 30 K filter (OD030C34, Cytiva) at 14,000g at 4 °C for 20 min. Pyrophosphate concentrations were measured in platelet-free plasma using ATP sulfurylase to convert pyrophosphate into ATP in the presence of excess adenosine 5’ phosphosulfate, followed by a luminescent assay of ATP, as described previously31,32.

Measurement of ALT, cytokines and chemokines

Assays were performed as previously described5. ALT (MAK052-1KT, Millipore Sigma) activity was measured according to the manufacturer’s instructions. The expression profile of cytokines and chemokines in mouse serum samples was determined using the BioLegend antivirus response panel (13-plex bead-based assay for the quantification of interferons (α, β, γ), interleukins (1β, 6, 10, 12p70) and chemokines (MCP-1, RANTES, CXCL-1, IP-10, TNF-α and GM-CSF)) according to the manufacturer’s instructions. Briefly, kit components were thawed and reconstituted as needed. Standards were prepared by 1:4 serial dilution in assay buffer. Serum samples were diluted 1:3 in Matrix A. Filter bottom plates were prewetted with wash buffer before the addition of standards and samples, then 25 µl of each standard and sample was added to the plates in duplicates and mixed with 25 µl of premixed beads solution. The plates were incubated with shaking at 500 r.p.m. for 2 h and washed twice, followed by the addition of 25 µl of detection antibodies and incubation at 500 r.p.m. for 1 h. Without washing, 25 µl streptavidin–phycoerythrin (SA-PE) was added to each well and incubated with shaking at 500 r.p.m. for 30 min. The plates were washed twice, and the beads were resuspended in 150 µl of wash buffer, transferred to fluorescence-activated cell sorting (FACS) tubes, and acquired on an BD LSR flow cytometer. The standard curve was used to determine the concentrations of each analyte in the serum samples using a five parameter logistic (5PL) fitting.

Data analysis

Sequencing data were analysed as described above. Other data were collected and analysed using GraphPad Prism v10.4.1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.