Nature： PERT tRNAs for disease-agnostic genome editing
Precise genome-editing technologies such as base editing1,2 and prime editing3 can
correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA.
Low copy number limits sup-tRNA efficiency
Low copy number limits sup-tRNA efficiency
The human genome encodes 47 isodecoder (anticodon) tRNA families comprising 418 high-confidence genes (Supplementary Table 1). This redundancy, coupled with transcription-induced mutagenesis, yields many tolerated tRNA mutations in humans, including anticodon mutations that create natural suppressor or mis-charged tRNAs25,26. Fourteen is odecoder families have no corresponding tRNA gene and are decoded by pseudocognate tRNAs27, and entire tRNA families have been deleted without consequence28. Therefore, we hypothesized that converting a single endogenous tRNA to a sup-tRNA with prime editing could be well-tolerated and enable readthrough of nonsense mutations regard less of the gene in which they originate. This approach avoids tRNA overexpression that can perturb global translation22–24 and preserves native tRNA regulation.
Previous sup-tRNA studies have heavily relied on high tRNA levels in transfection, LNP or viral settings, often using elevated copy numbers of the sup-tRNA and/or the PTC-containing mRNA4,5 . These findings revealed the need to identify sup-tRNAs that are sufficiently potent to mediate endogenous genomic PTC readthrough at low sup-tRNA
levels that are compatible with genomic installation and minimize the risk of perturbing global translation.
Screening prime editing-installed sup-tRNAs
Screening prime editing-installed sup-tRNAs
To comprehensively identify sup-tRNAs that promote stop codon readthrough with low copy number expression, we designed three prime editing screens to convert the anticodon of each endogenous human tRNA (n = 418 genes) to CUA, UCA, or UUA anticodons, thereby generating sup-tRNAs that recognize TAG, TGA or TAA stop codons,
respectively (Fig. 1a and Supplementary Fig. 2)
Whereas epegRNAs that simultaneously targeted multiple tRNA sequences generally performed best, half of the most enriched positive hits in the TAG screen were epegRNAs targeting a single tRNA (Extended Data Fig. 2b–d). Although the epegRNA screen successfully identified endogenous tRNAs that could be converted into effective sup-tRNAs,even the best-performing sup-tRNA from this screen achieved less than 5% protein yield relative to a wild-type GFP control (Extended Data Fig. 2b). To address this limitation, we next engineered all major features of each sup-tRNA to enhance read through efficiencies.
Exogenous promoter screen
Exogenous promoter screen
A eukaryotic tRNA gene contains three distinct elements: the leader sequence, the tRNA gene body and the termination sequence (Extended Data Fig. 4a). Whereas tRNA expression is typically driven by an internal Pol III promoter, the leader sequence is thought to regulate gene expression31. Thus far, most sup-tRNA studies have relied on exogenous
promoters for expression5 , although the role of internal tRNA promoters is being investigated32
Sup-tRNA leader and terminator sequences
Sup-tRNA leader and terminator sequences
The screen results also indicated that a terminator is essential for sup-tRNA activity, with a directly adjacent 5T terminator outperforming most endogenous terminator sequences (Extended Data Fig. 4g).We hypothesize that the absence of a terminator may lead to run-off of Pol III, or weakened expression due to a lack of the traditional Pol III recycling mechanism in which Pol III is rapidly reused for a new round of transcription after completing a transcript36. Notably, only 93% and 48% of natural downstream sequences have a 4T or 5T termination sequence, respectively, within 100 bp of their mature tRNA sequence37 (Supplementary Fig. 8a,b).
Saturation mutagenesis of sup-tRNAs
We next hypothesized that introducing mutations within the tRNA itself could further enhance sup-tRNA activity. We also sought to identify silent mutations that could improve prime editing out comes by evading cellular mismatch repair (MMR)38 and by prevent
ing rebinding of the prime editor to the target tRNA locus following repair. We selected three candidate sup-tRNAs (tRNA-Arg-CCT-4-1, tRNA-Tyr-GTA-2-1 and tRNA-Leu-TAA-4-1) and a corresponding mouse sup-tRNA (tRNA-Leu-TAA-2-1) and generated lentiviral libraries that include every possible 1-bp deletion, single-nucleotide substitution
variant (SNV), and simultaneous base pair substitution in each tRNA’s paired bases (Supplementary Fig. 2 and Supplementary Table 6).
Engineering an optimal Leu-TAA sup-tRNA
. We observed that the ac-only sup-tRNA and the engineered sup-tRNA are 5.6-fold and 2.5-fold less abundant, respectively, than the endogenous tRNA-Leu-TAA-1-1 that they replace (Extended Data Fig. 6a). Introducing mutations in the anticodon stem thus increased the abundance of the engineered sup-tRNA compared with the ac-only
sup-tRNA (Extended Data Fig. 6a). Both sup-tRNAs were modified at positions 26 and 37, as expected39 (Extended Data Fig. 6b–e). These findings suggest that mutations in the anticodon stem can increase engineered sup-tRNA performance in part by increasing its cellular abundance.
Optimization of sup-tRNA installation
Optimization of sup-tRNA installation
Encouraged by the greatly enhanced suppression efficiency achieved through iterative engineering of the mature tRNA sequence, we next optimized prime editing strategies to convert the mammalian endogenous tRNA-Leu-TAA-1-1 tRNA gene into the ac-only sup-tRNA or engineered sup-tRNA.
Off-target editing analysis
Off-target editing analysis
We observed no off-target prime editing or indel formation above background for all amplified genomic loci (Extended Data Fig. 8d).These data are consistent with previous reports of the high sequence specificity of prime editing, which probably arises from its mechanism requiring multiple independent DNA or RNA hybridization events in
order to make a productive edit3,42,43.
Effect of PERT on the transcriptome
Effect of PERT on the transcriptome
we evaluated the effect of repurposing the tRNA-Leu-TAA-1-1
tRNA gene on global tRNA homeostasis by performing quantitative PCR to measure the pooled expression of 28 different tRNA gene families following prime editing-mediated installation of an ac-only or engineered sup-tRNA (Leu-TAA-1-1+hp12ta>cg+hp13gc>cg). We observed no significant difference (adjusted P value > 0.05) in the expression
of any of these 28 tRNA gene families in either treatment condition relative to wild-type cells (Extended Data Fig. 8f), suggesting that the tRNA-Leu-TAA-1-1 tRNA gene can be repurposed without significant perturbation to general tRNA homeostasis.
Effect of PERT on the proteome
To evaluate potential readthrough of NTCs, we performed mass spectrometry to detect peptides that are translated into the 3′ UTR for protein-coding genes terminating with a TAG stop codon. After confirming that Leu-TAA sup-tRNAs retain 100% fidelity of leucine
charging (Extended Data Fig. 9a), we computationally predicted all peptides that could be generated by reading through the 4,036 natural TAG termination codons and extending translation to the second in-frame stop codon. We then examined whether these predicted pep tides were present by performing label-free quantitative mass spectrometry on trypsin-digested total protein lysates from both controland prime-edited HEK293T cells expressing and mCherry-STOP-GFP reporter and either the ac-only sup-tRNA or the engineered sup-tRNA (Leu-TAA-1-1+hp12ta>cg+hp13gc>cg).
Rescue of therapeutically relevant PTCs
Rescue of therapeutically relevant PTCs
Protein yields across the variants were moderately correlated (R = 0.49) with the readthrough scores measured in the pooled ClinVar screen (Supplementary Fig. 11c,d and
Supplementary Table 16), with a maximum observed protein yield of 14% relative to wild-type CFTR. Together, these results demonstrate that PERT can effectively rescue diverse pathogenic genomic PTCs and can be used to generate therapeutically relevant levels of full-length protein and active enzymes in human cells.
PERT in an in vivo reporter system
PERT in an in vivo reporter system
To assess the tolerability of PERT in vivo, we performed whole proteome mass spectrometry on tissues that had been prime edited to express an ac-only sup-tRNA. Similar to our observations in human cell lines (Fig. 4a,b), we did not detect any peptides corresponding to readthrough of natural stop codons in tissues from mice treated with
PERT (Supplementary Table 17). In addition, we monitored a separate cohort of mice over the course of 15 weeks and observed no significant difference in body weight in the PERT-treated mice (n = 7) relative to untreated controls (n = 9) (Extended Data Fig. 10b,c).
PERT in an in vivo Hurler syndrome model
Finally, we investigated the in vivo therapeutic application of PERT in a mouse model of the human lysosomal storage disease mucopolysac charidosis type I (MPS-I). MPS-1 is caused by a lack of α-L-iduronidase,leading to the pathogenic accumulation of glycosaminoglycans (GAGs).
This model harbours a TAG stop codon, Idua p.W392X, which is orthologous to the most common pathogenic human mutation, IDUA p.W402X,and has been used previously to evaluate PTC readthrough in vivo5,51.For this experiment we further engineered the mouse sup-tRNA using mutations identified via saturation mutagenesis screening of mouse tRNA-Leu-TAA-2-1, generating an engineered sup-tRNA (mouse tRNA-Leu-TAA-2-1+hp13gc>ta+mut38a>t) (Supplementary Fig. 15 and Supplementary Discussion).
Discussion
Discussion
Although direct DNA-level correction of pathogenic mutations remains the most straightforward approach to precision genetic medicine, the large effort currently required to develop, meet regulatory requirements and manufacture many gene editing agents needed to correct many mutations underscores the importance of complemen tary allele-agnostic and disease-agnostic approaches such as PERT to maximize the ability of patients to benefit from advances in gene editing. By enabling a single treatment to address a broad cross-section of nonsense mutation-related diseases, PERT represents an advance
toward making therapeutic genome editing accessible to a much larger patient population. We hope that PERT and the flexibility of prime editing will stimulate additional allele-agnostic and mutation-agnostic therapeutic gene editing strategies that may provide broadly applicable treatments for diverse serious disorders.
标签：
PERT
