From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson’s disease or amyotrophic lateral sclerosis progress and can be treated.

Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells.

To remove this obstacle, Li et al. focused on prime editing, a cutting-edge ‘search and replace’ approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases.

A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson’s disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form.

The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

One of the current challenges of using human pluripotent stem cells (hPSCs) to model human diseases is to precisely and efficiently engineer the genome to introduce designer mutations (Hockemeyer and Jaenisch, 2016; Soldner and Jaenisch, 2018). Currently, the predominant approach in hPSCs is to induce targeted DNA double-strand breaks (DSBs) using highly active site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system (Cong et al., 2013; Ding et al., 2013; Jinek et al., 2012; Jinek et al., 2013; Mali et al., 2013) or protein engineering platforms including zinc finger nucleases (Hockemeyer et al., 2009; Soldner et al., 2011; Zou et al., 2009) and transcription activator-like effector nucleases (TALEN) (Boch et al., 2009; Cermak et al., 2011; Hockemeyer et al., 2011). Such targeted DSBs have been shown to substantially increase genome editing efficiency over conventional homologous recombination. However, since nuclease-induced DSBs are in most contexts preferentially repaired by non-homologous end joining compared with homology-directed repair (HDR) mechanisms, DSB-mediated genome editing frequently generates undesirable compound heterozygous editing outcomes with one correctly targeted allele and insertion or deletion (indel) on the second allele, causing the disruption of the protein-coding sequence (Cox et al., 2015). Therefore, it has been challenging to generate disease-associated dominant mutations in a heterozygous setting. By contrast, PE has been shown to overcome this limitation in a wide variety of cell types, as it does not require a DSB but directly repairs a nicked DNA strand (Anzalone et al., 2019). PE is a multicomponent editing system composed of a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended prime editing (PE) guide RNA (pegRNA) that is reverse transcribed and functions as a repair template at the target site (Anzalone et al., 2019). While successful PE has been previously demonstrated in hPSCs (Chemello et al., 2021; Chen et al., 2021; Sürün et al., 2020), it remains unclear whether this approach has the potential to facilitate the generation of hPSC-based disease models. Here, we systematically compare different genome editing methods and show that PE is overall more efficient and precise to introduce heterozygous point mutations into hPSCs. Furthermore, by optimizing the delivery modality of the PE components, we were able to establish a highly efficient genome editing platform for hPSCs. By comparing plasmid, ribonucleoprotein (RNP), and in vitro transcribed mRNA delivery, we found that nucleofecting nCas9-RT as mRNA combined with synthetically generated and chemically modified pegRNAs yielded editing efficiencies exceeding 60%, which is comparable to efficiencies observed in tumor cell lines (Anzalone et al., 2019; Nelson et al., 2021). Together, these data indicate that PE has the potential to become the preferred method for genome editing of hPSCs.

To evaluate the potential use of PE to genetically modify hPSCs, we directly compared editing outcomes of PE to established CRISPR/Cas9 and TALEN targeting approaches with the goal of introducing disease-relevant point mutations. Initially, we chose to target the leucine rich repeat kinase 2 (LRRK2) gene to introduce the G2019S (G6055A) mutation (Gilks et al., 2005), which is one of the most frequent pathogenic substitutions linked to Parkinson’s disease (PD). This mutation is found in approximately 4% of dominantly inherited familial PD cases, in both heterozygous and homozygous forms, and around 1% of sporadic PD cases (Healy et al., 2008). To introduce the G2019S (G6055A) mutation into hPSCs, we generated plasmid-based CRISPR/Cas9, TALEN and PE reagents (without [PE2] or with secondary nicking guide RNAs [ngRNA; PE3]) using previously established optimized design and targeting procedures (Figure 1A; Anzalone et al., 2019; Hockemeyer et al., 2011; Hsu et al., 2021; Soldner et al., 2011; Soldner et al., 2016). Briefly, we co-electroporated the human embryonic stem cell (hESC) line WIBR3 with the respective genome engineering components and an enhanced green fluorescent protein (EGFP)-expressing plasmid to allow for the enrichment of transfected cells by fluorescence activated cell sorting (FACS). Genome editing outcomes were evaluated in the transfected bulk cell population (EGFP-positive) 72 hr after electroporation and in single cell-derived subclones. Next generation sequencing (NGS) of amplicons spanning the G2019S (G6055A) region indicated that 2–4% of alleles carried the correct G2019S (G6055A) substitution (Figure 1B and Figure 1—figure supplement 1A). While we observed roughly comparable editing efficiencies using CRISPR/Cas9, TALEN, and PE (with secondary ngRNA [PE3]) gene targeting, all HDR-based approaches generated a significantly higher number of undesired editing outcomes with 19.6 and 3.3% indels for CRISPR/Cas9 and TALEN, respectively, compared with less than 0.5% for PE (Figure 1B and Figure 1—figure supplement 1A). Genotyping of expanded single cell-derived clones showed higher efficiency in generating heterozygous correctly targeted cell lines using PE primarily due to a substantially higher number of compound heterozygous editing outcomes for CRISPR/Cas9 and TALEN targeting with the correctly inserted G6055A sequence variant on one allele and indels on the second allele (Figure 1C, as identified by restriction fragment length polymorphism [RFLP] and Sanger sequencing analysis). Together, these data indicate that PE is overall more efficient and substantially more precise in generating heterozygous mutations in hPSCs, as compared with traditional DSB-based genome engineering approaches.

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To scale the PE-based genome editing approach and streamline the derivation of correctly modified single cell clones, we applied a recently established genome editing platform, which employs multiplex low cell number nucleofection, limited dilution, and NGS-dependent genotyping instead of the time-consuming and laborious FACS sorting and manual single cell expansion steps to isolate correctly edited hPSC lines (see Figure 1—figure supplement 2 and Experimental procedures for details). While this approach results in slightly lower overall bulk editing efficiencies (as determined by NGS), most likely due to the lack of FACS-based enrichment of transfected cells, the substantially reduced number of cells required and the streamlined workflow allows for highly efficient, multiplexed generation of genome-edited hPSC lines in parallel in less than 4 weeks (Figure 1—figure supplement 2). Importantly for this work, the omission of FACS enrichment allowed us to systematically and simultaneously compare a larger number of delivery modalities, as described below.

To confirm the feasibility of using PE to introduce point mutations efficiently and robustly into hPSCs, we tested PE at additional genomic loci. We were able to introduce mutations into the previously published and commonly targeted HEK site 3 (HEK3) locus (CTT-sequence insertion) (Anzalone et al., 2019), as well as two additional PD-associated mutations into the α-Synuclein (SNCA) locus (A30P [G88C]; Krüger et al., 1998; and A53T [G209A] Polymeropoulos et al., 1997; Figure 1—figure supplement 1B, C) with editing efficiencies comparable to the LRRK2 locus (Figure 1D and E; quantified as pure prime editing efficiencies [PPE] as defined in Petri et al., 2021). As described for LRRK2, the analysis of single cell-expanded clones revealed the efficient generation of heterozygous and homozygous hPSC lines carrying the dominant A30P mutation in the SNCA gene (Figure 1—figure supplement 3A, B). Importantly, representative cytogenetic analysis of single cell-expanded LRRK2 (G2019S) and SNCA (A30P) clones showed normal karyotypes for seven out of seven tested cell lines. Together, these experiments demonstrate that PE can be used to robustly and efficiently introduce disease-associated mutations into hPSCs to generate isogenic disease models.

During these experiments, we noted that editing outcomes for both the PE2 and PE3 approaches appeared considerably lower than what was previously reported for a variety of human tumor cell lines (Anzalone et al., 2019; Nelson et al., 2021). Indeed, we found that plasmid-based targeting of the HEK3 (CTT insertion) locus resulted in only ~4.3% PPE in WIBR3 hESCs compared with ~12.7% PPE in HEK293T tumor cells using the PE3 strategy (Figure 1E). Similar differences in gene editing efficiencies between hPSCs, primary cells, and tumor cell lines have been commonly observed for other genome engineering approaches including CRISPR/Cas9 targeting (Bowden et al., 2020; Haapaniemi et al., 2018; Ihry et al., 2018). It remains unclear if this difference is the result of cell-intrinsic factors that restrict genome editing specifically in hPSCs or whether the low efficiency is a consequence of insufficient delivery of the PE components.

To estimate to which extend PE efficiencies could be increased in WIBR3 hESCs by optimized delivery of the prime editor, we used a recently described approach (Bharucha et al., 2021; Habib et al., 2022) and expressed the nCas9-RT protein (PE2 version of the prime editor protein as described in Anzalone et al., 2019) followed by a 2A-EGFP fluorescent reporter from the AAVS1 safe harbor locus (DeKelver et al., 2010; Hockemeyer et al., 2009; Figure 2A). We established an hPSC clone that uniformly expressed GFP and maintained pluripotency (Figure 2B and Figure 2—figure supplement 1). Nucleofection of these cells with a plasmid encoding the HEK3 (CTT insertion) pegRNA (without [PE2] or with [PE3] secondary ngRNA) or with a chemically-modified synthetic pegRNA (without [PE2] or with [PE3] secondary ngRNA) resulted in substantially increased editing efficiencies of up to 22% of correctly inserted modifications (Figure 2C). Similarly, targeting the LRRK2 (G2019S) and SNCA (A30P) loci with chemically-modified synthetic pegRNAs (without [PE2] or with [PE3] secondary ngRNA) resulted in editing efficiencies up to 12% and 27%, respectively (Figure 2D). While these data do not exclude fundamental biological differences in the PE process between hPSCs and other cell types, these experiments demonstrate that the method of delivery of the PE components has a significant role in dictating the genome editing efficiency in hPSCs, which can be comparable to the efficiencies observed in tumor cell lines and primary cells.

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To improve PE efficiencies using transient delivery of the PE components, we set out to optimize PE delivery conditions. Initially, we focused on delivering the PE components as RNPs, a highly efficient approach described for CRISPR/Cas9-mediated genome editing (Zuris et al., 2015), which was recently successfully adapted for PE in zebrafish and human primary T cells (Petri et al., 2021). Using recombinant nCas9-RT protein (PE2 version of the prime editor protein as described in Anzalone et al., 2019 purified from bacteria; Figure 2—figure supplement 2A) and the previously established protocols for RNP-based CRISPR/Cas9 editing (Petri et al., 2021; Zuris et al., 2015), we nucleofected pre-assembled RNPs containing the recombinant nCas9-RT protein and chemically modified synthetic pegRNAs (without [PE2] or with [PE3] secondary ngRNA) targeting the HEK3 (CTT insertion), LRRK2 (G2019S), and SNCA (A30P) loci. Consistent with previous reports on other cell types, we observed RNP-mediated editing outcomes in hPSCs with locus-dependent efficiencies between 1 and 6% (Figure 2E and Figure 2—figure supplement 2B and Figure 2—figure supplement 2C). While these data clearly indicate the feasibility of RNP-based PE in hPSCs, the observed efficiencies are comparable to the plasmid-based approach and far below the efficiencies observed with stable expression of nCas9-RT from the AAVS1 locus. To exclude that RNP-based PE efficiencies were concentration- or batch-dependent, we repeated some of these experiments with protein from independently purified nCas9-RT batches (Figure 2—figure supplement 2D) and used higher protein concentrations (Figure 2—figure supplement 2E). However, none of these conditions resulted in substantially improved RNP-based editing efficiencies in hPSCs.

An alternative approach, allowing highly efficient delivery of Cas9 for CRISPR/Cas9-based genome editing (Chang et al., 2013; Hwang et al., 2013; Wang et al., 2013), is to deliver the prime editor using in vitro transcribed mRNA (Chen et al., 2021; Sürün et al., 2020). To systematically compare plasmid-, RNP-, and mRNA-based PE at the above-established HEK3 (CTT insertion), LRRK2 (G2019S), and SNCA (A30P) loci, we nucleofected either: (i) plasmid-based nCas9-RT together with plasmid-based pegRNAs (without [PE2] or with [PE3] secondary ngRNA), (ii) preassembled RNPs containing nCas9-RT protein and chemically modified synthetic pegRNAs (without [PE2] or with [PE3] secondary ngRNA), or (iii) in vitro transcribed nCas9-RT mRNA together with chemically modified synthetic pegRNAs (without [PE2] or with [PE3] secondary ngRNA). Bulk NGS revealed that the combination of in vitro transcribed mRNA-based delivery of the nCas9-RT with chemically modified synthetic pegRNAs and ngRNAs consistently increased editing efficiencies across all three tested loci up to 13-fold compared with plasmid- and 8-fold compared with RNP-delivery (Figure 3A). Using this optimized in vitro transcribed mRNA-based delivery approach allowed us to achieve editing efficiencies up to 26.7% (for the SNCA locus). When combined with secondary nicking of the non-edited strand (PE3), these editing efficiencies are similar to using the stably nCas9-RT expressing cell lines and comparable to efficiencies commonly observed in human tumor cell lines (Anzalone et al., 2019; Nelson et al., 2021). While mRNA-based editing efficiencies seem to depend on the approach used to in vitro transcribe the nCas9-RT mRNA (Figure 3—figure supplement 1A), we found that mRNA-based editing efficiencies are highly consistent across different nCas9-RT mRNA batches when using the best in vitro transcription conditions (Figure 3—figure supplement 1B). Furthermore, we observed that using single-stranded mRNA compared with either RNPs or double-stranded plasmid DNAs resulted in improved overall health and increased survival of single cells as indicated by increased clonal survival following nucleofection (Figure 3B). To test if the high efficiency of mRNA-based PE is a unique feature of the WIBR3 hESCs, we repeated some key experiments in a second human-induced pluripotent stem cell (hiPSC) line 8858 (Paşca et al., 2015) by targeting the LRRK2 (G2019S) and SNCA (A30P) loci and found comparable editing efficiencies (Figure 3C). Importantly, we were able to establish single cell-derived clones carrying the correct SNCA (A30P) and LRRK2 (G2019S) mutations with high efficiency (Figure 3D). Considering that potential therapeutic applications would require precise genome editing of hPSCs in xeno-free conditions, we were able to show efficient and robust PE of the LRRK2 (G2019S) locus in WIBR3 hESCs using several commonly used feeder-free culture systems with comparably high PE efficiencies (Figure 3—figure supplement 1C).

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A major limitation of classical CRISPR/Cas9 targeting remains the high number and complexity of undesirable editing outcomes (indels). These alleles are resistant to targeting with the same reagents and thus limit the overall HR editing efficiencies in the context of continued editing or retargeting. Given the much reduced occurrence of indel-containing alleles in mRNA-based PE, we hypothesized that this approach might allow efficient retargeting of the same locus. Indeed, we find for all tested loci (HEK3, LRRK2, and SNCA) that additional rounds of mRNA-based PE of the same cell population could substantially increase overall editing efficiencies (Figure 4A). This indicates that multiple rounds of mRNA-based PE could result in precise and nearly complete editing of a bulk hPSC population without any type of selection.

Figure 4

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The data presented thus far describes the insertion of disease-associated mutations into a wild-type genetic background. To test whether mRNA-based PE can be used to correct disease-causing mutations, we designed pegRNAs to specifically target only the mutated SNCA (A30P) allele to revert this mutation back to wild type in hPSCs. When targeting heterozygous (Figure 4B and C) or homozygous (Figure 4D and E) SNCA (A30P) hESC lines, bulk NGS indicated the correction of 31.0 (Figure 4B) and 30.1% (Figure 4D) of the mutated A30P alleles, respectively. Subsequent genotyping of single-cell derived clones indicated 26.2 (Figure 4C) and 48% (Figure 4D; combined heterozygous and homozygous) precisely corrected clones without additional undesired modifications (indels) of the wild-type allele. Taken together, our data indicate that in vitro transcribed mRNA-based PE is a highly efficient gene editing approach in hPSCs that has the potential to greatly facilitate the generation of disease-specific hPSC models.

The experiments performed here provide a detailed experimental road map for how to implement PE towards genome engineering of hPSCs. We show that mRNA transfection of the prime editor component (nCas9-RT) paired with the transfection of chemically modified guide RNAs is well tolerated and highly effective for introducing precise designer mutations in hiPSCs and hESCs. This work focuses on PE, which is highly versatile to introduce not only a wide range of disease-associated single nucleotide sequence variants but also more complex genetic alterations such as insertions and deletions. However, there are additional non-DSB-based genome editing approaches (e.g. based editors) which, dependent on the specific context, have been shown to efficiently introduce genetic modifications in hPSCs (we refer to recent reviews for a detailed discussion of the advantages and disadvantages of such approaches Anzalone et al., 2020; Molla et al., 2021; Zeballos C and Gaj, 2021). Although not tested, we believe that the here described RNA transfection-based delivery modalities could be adapted to increase genome editing efficiencies for genome editing approaches other than PE. Considering that mRNA-based PE does not require specialized molecular or biochemical skills and consistently achieves high editing efficiency in hESC and hiPSC lines, we predict that this approach has the potential to greatly facilitate the generation of disease-specific hPSC models and will be widely adopted by researchers.

During the process of establishing this workflow, we made several key observations. We find that PE can be as efficient in hPSCs as has been reported for cancer cells (Anzalone et al., 2019; Nelson et al., 2021). We demonstrate that this approach efficiently allows for the introduction or correction of heterozygous disease-related mutations in hPSCs with base-pair precision and without introducing undesired additional modifications on the second allele. The resulting cells showed a normal karyotype, consistent with low genotoxicity of PE due to the lack of DSBs (Anzalone et al., 2019).

A recent study reported comparable high PE efficiencies in a doxycycline-inducible PE2-expressing hESC line (Habib et al., 2022). The authors observe that PE3-mediated PE is generally accompanied by the generation of indels around the target site caused by the combinatory activity of the RT and pegRNA. While we also find a certain degree of indels (impure PE [IPE] and by-products) at the target site using the PE3 approach, the frequency is usually low compared with the intended sequence modifications which is consistent with previous data in other cell types (Anzalone et al., 2019; Chen et al., 2021; Nelson et al., 2021). While it has been shown that the frequency of undesired edits varies widely across genomic loci and is pegRNA dependent, it is possible that indel frequency is also affected by the different delivery modalities (e.g. different mRNA transfection of the PE component paired with the transfection of chemically modified guide RNAs has different kinetics and expression levels compared with other approaches). Due to the unique property of hPSCs to allow for the expansion of clonal cell lines, we do not believe that such on-target indels limit the use of PE for disease modeling approaches because undesired modifications can be easily excluded through targeted sequencing during quality control of individual clones.

In the past, generating such isogenic cell lines that differ exclusively at individual disease-causing sequence variants was highly laborious and an experimental bottleneck. Here we overcome this challenge by deploying PE via optimized delivery methods. We demonstrate that hPSCs can be subjected to several rounds of PE, eventually yielding up to 60% correctly targeted alleles. Importantly, PE efficiencies might be further increased by including mRNAs coding for DNA mismatch repair inhibiting proteins, a novel approach that has been recently shown to significantly improve the PE platform (Chen et al., 2021). These very high editing efficiencies without the need for selection of enrichment of targeted clones provide an intriguing platform to develop more robust in vitro disease models and potential therapeutic applications of PE in hPSCs or differentiated cell types. In our study, we successfully introduced three out of three familial PD point mutations into hPSCs using previously established algorithms to design pegRNAs (Hsu et al., 2021). In each case, a classical protospacer adjacent motif (PAM) was present close to the intended amino acid substitution and we did not explore more complex or challenging genetic modifications. As is the case for all genome editing approaches, PE efficiencies vary widely depending on the specific genomic context and pegRNA design, and certain genetic modifications will require more extensive pegRNA testing and validation. However, we expect systematic approaches that establish optimized design parameters for PE, as recently described for cancer cells (Kim et al., 2020b; Nelson et al., 2021) and the development of Cas9 variants with non-classical PAMs (Chatterjee et al., 2020; Kleinstiver et al., 2015; Miller et al., 2020) will overcome these limitations and combined with the optimized protocols reported here will allow PE to become a general method of choice for genome editing in hPSCs.

The focus of this work was to establish a highly efficient platform for PE in hPSCs and although all generated prime edited cell lines showed a normal karyotype consistent with low genotoxicity of PE due to the lack of DSBs (Anzalone et al., 2019), it is important to point out that we refrained from a detailed off-target analysis. We believe that there are already available substantial datasets in a variety of cell types indicating that PE is highly specific and shows much lower guide RNA-dependent or independent off-target effects compared with DSB-based CRISPR/Cas9 or base editing approaches (Gao et al., 2021; Geurts et al., 2021; Habib et al., 2022; Jin et al., 2021; Kim et al., 2020a; Schene et al., 2020). Genome-wide off-target analyses of in vitro generated cell lines including whole genome sequencing-based approaches remain challenging even for conventional CRISPR/Cas9-based cutting approaches due to the substantial number of genetic alterations which occur during regular cell culture (Kuijk et al., 2020). Thus, a key future step toward the development of clinical PE approaches will require the development of sophisticated off-target analyses tools that account for nCas9 single strand break-mediated and transient reverse transcriptase expression-mediated genetic alterations.

Sequencing data can be accessed through the repository platform Zenodo (https://doi.org/10.5281/zenodo.6941502). The datasets for AAVS1 knock-in genotyping, aCGH karyotyping, and the source data files used to generate the featured graphs and tables can be found on Zenodo (https://doi.org/10.5281/zenodo.6941599). Plasmids referred to in this paper have been deposited to Addgene's Michael J. Fox Foundation Plasmid Resource and their associated RRID can be found in Supplemental table 2.

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Author details

1. Hanqin Li

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
   3. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Oriol Busquets

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7995-1084

2. Oriol Busquets

   1. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States
   2. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, The Bronx, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Hanqin Li

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1372-9699

3. Yogendra Verma

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Khaja Mohieddin Syed

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Nitzan Kutnowski

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3012-4616

6. Gabriella R Pangilinan

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

7. Luke A Gilbert

   1. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States
   2. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, United States
   3. Department of Urology, University of California, San Francisco, San Francisco, United States
   4. Arc Institute, Palo Alto, United States

   Contribution

   Conceptualization, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

8. Helen S Bateup

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States
   3. Chan Zuckerberg Biohub, San Francisco, United States
   4. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States

   Contribution

   Supervision, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0135-0972

9. Donald C Rio

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing – review and editing

   For correspondence

   don_rio@berkeley.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4775-3515

10. Dirk Hockemeyer

    1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    2. Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
    3. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States
    4. Chan Zuckerberg Biohub, San Francisco, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    hockemeyer@berkeley.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5598-5092

11. Frank Soldner

    1. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, United States
    2. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, The Bronx, United States
    3. Department of Genetics, Albert Einstein College of Medicine, The Bronx, United States
    4. Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, The Bronx, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    frank.soldner@einsteinmed.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7102-8655

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