Author response: Highly efficient generation of isogenic pluripotent stem cell models using prime editing
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson’s disease in hPSCs. Editor's evaluation In this manuscript, Li et al., directly compare different editing strategies for human pluripotent stem cells and demonstrate that prime editing is more efficient and precise, compared with double strand break-based methods. They also confirm the suitability of prime editing for the introduction of different mutations related to Parkinson’s disease as a model. In this process the authors noted a lower editing efficiency of human pluripotent stem cells, compared with tumour cell lines, and explored ways to improve it. Nucleofection of in vitro-transcribed mRNA-based delivery approach significantly increased the editing efficiency, without the need to select for targeted clones, and multiple rounds of mRNA-based prime editing could yield near complete editing of hPSCs, including disease-causing mutations. The proposed platform paves the way for future prime editing methods for hPSCs. https://doi.org/10.7554/eLife.79208.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest 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. Introduction 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. Results 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. Figure 1 with 3 supplements see all Download asset Open asset Systematic comparison of CRISPR/Cas9, transcription activator-like effector nucleases (TALEN), and prime editing (PE)-based genome editing efficiencies in human embryonic stem cells (hESCs) using plasmid-based delivery. (A) Schematic depicting genome editing strategies to generate the leucine rich repeat kinase 2 (LRRK2; G2019S[G6055A]) mutation. Exon, gray shade; prospacers for CRISPR/Cas9 and PE, pink boxes; protospacer adjacent motif (PAM) sequences, purple boxes; representative PE guide RNA (pegRNA) extension, green arrow; TALEN recognition sites, blue boxes; single-stranded oligodeoxyribonucleotide (ssODN) homology-directed repair (HDR) template for CRISPR/Cas9 targeting, yellow box; the nucleotide to mutate, black open square; intended mutation, red filled square. (B) Comparison of bulk genome editing outcomes between CRISPR/Cas9, TALEN, and PE to insert the LRRK2 (G2019S) mutation (aggregated analysis using four different pegRNA designs and two TALEN pairs), N=2 for CRISPR/Cas9, N=3 for TALEN, N=2 for PE2, and N=6 for PE3. For individual analysis see Figure 1—figure supplement 1. (C) A summary of clonal genotyping comparing different genome editing strategies indicating the number and editing efficiency of single cell-derived clones carrying the correct heterozygous (HET) or homozygous (HOMO) G2019S substitution as identified by restriction fragment length polymorphism analysis (RLFP) followed by Sanger sequencing to excluded additional insertions or deletions (indels). (D) Generating α-Synuclein (SNCA) familiar Parkinson’s disease (PD) mutations, A30P, and A53T, using PE. pure prime editing (PPE), precise PE; IPE, impure PE; by-product, other indels. A30P, N=3; A53T, N=2. (E) Comparison of bulk PE outcomes on HEK3 (CTT-insertion) edits between hESCs and HEK293T cells. Color scheme, same as (D). N=3. (Error bars indicate the SD, N=number of biological replicates). 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. Figure 2 with 2 supplements see all Download asset Open asset Prime editing (PE) in human embryonic stem cells (hESCs) expressing Cas9-nickase fused to a reverse transcriptase (nCas9-RT) protein from the AAVS1 safe harbor or delivered as RNP. (A) Schematic of the genome editing strategy to knock-in nCas9-RT-2A-GFP (PE2 version of the prime editor protein as described in Anzalone et al., 2019) into the AAVS1 locus. (B) Expression of green fluorescent protein (GFP) and immunostaining of OCT4 on hESCs with nCas9-RT-2A-GFP knock-in. Scale bar = 100 µm. (C) Comparison of bulk PE outcomes between plasmid-expressed PE guide RNAs (pegRNAs)/nicking guide RNAs (ngRNAs) and synthetic pegRNAs/ngRNAs on HEK3 (CTT-insertion) edits in hESCs with nCas9-RT-2A-GFP knock-in. N=3. (D) Bulk PE outcomes on leucine rich repeat kinase 2 (LRRK2; G2019S) and α-Synuclein (SNCA; A30P) edits using synthetic pegRNAs/ngRNAs in hESCs with nCas9-RT-2A-GFP knock-in. N=3. (E) Bulk PE outcomes from RNP delivery on HEK3 (CTT-insertion), LRRK2 (G2019S), and SNCA (A30P) edits in human pluripotent stem cells (hPSCs). N=6. (Error bars indicate the SD, N=number of biological replicates). 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 we repeated some experiments in a second pluripotent stem cell line et al., by targeting the LRRK2 (G2019S) and SNCA (A30P) loci and found comparable editing efficiencies (Figure Importantly, we were able to establish single cell-derived clones carrying the correct SNCA (A30P) and LRRK2 (G2019S) mutations with high efficiency (Figure that potential require precise genome editing of hPSCs in we were able to show efficient and PE of the LRRK2 (G2019S) locus in WIBR3 hESCs using commonly used with high PE efficiencies (Figure 3—figure supplement Figure 3 with 1 supplement see all Download asset Open asset efficient prime editing (PE) in human pluripotent stem cells (hPSCs) using mRNA-based delivery. (A) Comparison of bulk PE outcomes between plasmid, and mRNA-based delivery on indicated modifications in mRNA N=3; RNP data shown in Figure 2E was in this analysis for N=6. (B) and of comparing of hESCs after nucleofection between plasmid, and mRNA-based delivery. N=2. (C) Bulk PE outcomes on leucine rich repeat kinase 2 (LRRK2; G2019S) and α-Synuclein (SNCA; A30P) edits in a pluripotent stem cells line using mRNA-based delivery. N=2. (D) A summary of clonal genotyping from LRRK2 (G2019S) and SNCA (A30P) PE in indicating the number and editing efficiency of single cell-derived clones carrying the correct heterozygous (HET) or homozygous (HOMO) of biological bars indicate the for A major limitation of CRISPR/Cas9 targeting remains the high number and of undesirable editing outcomes (indels). alleles are to targeting with the same reagents and limit the overall editing efficiencies in the of editing or the reduced of alleles in mRNA-based PE, we that this approach allow efficient of the same locus. Indeed, we for all tested loci LRRK2, and that additional rounds of mRNA-based PE of the same cell population could substantially increase overall editing efficiencies (Figure This that multiple rounds of mRNA-based PE could result in precise and complete editing of a bulk hPSC population without of Figure 4 Download asset Open asset prime editing (PE) and of an α-Synuclein (SNCA; A30P) mutation in human embryonic stem cells (A) Comparison of bulk PE outcomes in a strategy using mRNA-based delivery on the three indicated mutations in N=3. (B) Bulk generation sequencing (NGS) analysis indicating allele and after mRNA-based PE single to correct the heterozygous SNCA (A30P) mutation in N=3. The line the allele The of the allele from the allele is (C) and summary of clonal genotyping from an SNCA (A30P) heterozygous mutation in a The with edited clones containing more than insertion or deletion (indel) were as without (D) Bulk NGS analysis indicating allele and after mRNA-based PE single to correct the homozygous SNCA (A30P) mutation in N=3. (E) and summary of clonal genotyping from an SNCA (A30P) homozygous mutation in a The with edited clones containing more than were as without (Error bars indicate the SD, N=number of biological replicates). The data far the insertion of disease-associated mutations into a genetic To test whether mRNA-based PE can be used to correct disease-causing mutations, we pegRNAs to specifically target only the mutated SNCA (A30P) allele to revert this mutation back to in hPSCs. When targeting heterozygous (Figure and C) or homozygous (Figure and SNCA (A30P) lines, bulk NGS indicated the of (Figure and (Figure of the mutated A30P genotyping of clones indicated (Figure and (Figure combined heterozygous and precisely clones without additional undesired modifications of the our data indicate that in vitro transcribed mRNA-based PE is a highly efficient gene editing approach in hPSCs that has the potential to facilitate the generation of hPSC models. Discussion The experiments a for how to PE genome engineering of hPSCs. We show that mRNA of the prime editor (nCas9-RT) with the of chemically modified guide RNAs is well and highly effective for introducing precise designer mutations in and This on PE, which is highly to introduce not only a wide of disease-associated single nucleotide sequence but also more genetic such as insertions and However, are additional genome editing approaches on the specific have been shown to efficiently introduce genetic modifications in hPSCs to recent for a of the and of such approaches Anzalone et al., 2020; et al., 2021; and 2021). not we that the described RNA delivery modalities could be adapted to increase genome editing efficiencies for genome editing approaches other than PE. that mRNA-based PE does not require or and consistently high editing efficiency in and lines, we that this approach has the potential to facilitate the generation of hPSC models and be by During the process of this we We 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 of heterozygous mutations in hPSCs with precision and without introducing undesired additional modifications on the second The cells showed a normal consistent with low of PE due to the lack of DSBs (Anzalone et al., 2019). A recent study reported comparable high PE efficiencies in a line et al., The authors that PE is by the generation of indels around the target site by the of the
Source: eLife
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
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OpenAlex ID: W4312301237