A molecule sensor using a two-part designed guide RNA

To achieve a molecular proximity sensor that drives genome editing, a specific physical interaction between two molecules needs to be converted into a successful genome editing event. One possibility is to ‘split’ a key molecule into two parts, but in such a way that bringing complementary non-functional parts into proximity restores its molecular function. This strategy has been successful in various protein designs (Michnick et al., 2007), such as split-GFP (Ghosh et al., 2000), split-Luciferase (Kim et al., 2004), or split-Protease (Wehr et al., 2006) to convert protein-protein interactions or proximities into various output signals based on fluorescence, bioluminescence, or protein modification. In the split architecture, the ‘dimerization module’ is a key sensor component. Although strategies that split the protein component of the genome editing complex have been described (e.g. split-Cas9 [Yu et al., 2020]), we reasoned that having the gRNA serve as the dimerization module rather than the protein, i.e., by splitting it into two parts, and making the restoration of its function dependent on a molecular proximity event, would afford even more control. For example, if multiple split gRNAs were present within the same cell, they could be independently controlled, whereas a split Cas9 would only allow a single control point. In our initial experiments, we focused on splitting the pegRNA used in prime editing.

We considered three broad strategies in designing a ‘split-pegRNA’ system. First, the pegRNA could be split into a functional sgRNA and its 3’-extension sequence containing the reverse-transcription template and primer binding sequence, the latter also referred to as prime editing trans RNA or petRNA by a recent report (Liu et al., 2022; Figure 1a, top). Second, an extra sequence motif such as a self-splicing split-ribozyme could be inserted within the pegRNA sequence, such that the coming together of split-ribozyme parts would be required to splice out the extraneous sequence and yield a functional pegRNA (Figure 1a, middle). Third, the pegRNA could be divided at the repeat:anti-repeat junction, which was originally joined with a GAAA RNA tetraloop to form a single-guide RNA (sgRNA) molecule from crRNA and tracrRNA (Jinek et al., 2012; Figure 1a, bottom).

We first tested splitting the pegRNA into a functional sgRNA and petRNA (Figure 1a, top; Figure 1—figure supplement 1a–b). We reasoned that the addition of complementary RNA sequences as a ‘dimerization module’ at the split junction of sgRNA and petRNA would drive the formation of an active pegRNA complex only when the correct complementary sequences are present. To inhibit early degradation of split RNAs, we appended an additional RNA pseudoknot structure at the ends of both molecules, borrowing from the strategy used to form the enhanced pegRNA or ‘epegRNA’ for higher prime editing efficiency (Nelson et al., 2022). We tested a handful of RNA:RNA dimerization sequences that range from 13 to 164 base-pairs, with an expectation that base-pairing between dimerization sequences would drive the formation of active pegRNA molecules. To measure the editing efficiency of the sgRNA:petRNA splitting strategy, we cloned each RNA in separate RNA expression vectors. We programmed crRNA:petracrRNA to target the endogenous HEK3 locus and insert CTT at the +0 position relative to the nick in combination with the ‘PE4max’ prime editor (also referred to as PEmax-P2A-hMLH1dn), as this edit has been previously used to benchmark prime editing (Anzalone et al., 2019; Chen et al., 2021). In general, we observed a low editing efficiency (<2%) by sgRNA-petRNA pairs that are expected to be dimerized by complementary RNA sequences, even with an additional 3’ RNA pseudoknot structure that inhibits RNA degradation from 3’-end (Nelson et al., 2022; Figure 1—figure supplement 1c). The inefficiency in editing is unlikely to be due to the inserted dimerization domain because a single pegRNA with additional RNA stem-loop structure at the PE-junction exhibited moderate editing efficiency (~10%; pegRNA w/ SL condition in Figure 1—figure supplement 1c). The underlying cause might be the inefficient dimerization driven by RNA-RNA duplex formation in the cellular environment without additional protein binding to the RNA duplex, or possible degradation of RNA duplex that lies outside of the Cas9-gRNA complex, unprotected from other factors for binding.

Second, we tested the idea of inserting self-splicing ribozymes within the pegRNA (Figure 1a, middle; Figure 1—figure supplement 2a). A potential advantage of this strategy is that splicing out of the ribozyme sequence would result in an identical molecule to the standard pegRNA. We tested six sites within pegRNA to insert the whole self-splicing ribozyme sequence from Tetrahymena thermophila (413 bp in length) (Herschlag and Cech, 1990; Figure 1—figure supplement 2b). If this proved successful, we envisioned that the ribozyme could be split into two parts and function as a ‘split-ribozyme’ (Gambill et al., 2022). However, editing using pegRNAs containing the self-splicing ribozyme sequence was inefficient (<2%) in all six constructs. To the limited extent that editing was observed, we found that it was dependent on ribozyme function because including a loss-of-function substitution in the ribozyme sequence reduced editing efficiency by greater than 10-fold (Figure 1—figure supplement 2c). Our results suggest that while prime editing depends on the self-splicing function of the ribozyme to produce an active pegRNA, the overall ribozyme efficiency is too low, possibly due to the presence of a prime editing enzyme that might bind and interfere with self-splicing.

Finally, we tried splitting the pegRNA into a crRNA component and a tracrRNA component with the 3’-extension necessary for prime editing (we term the latter as the prime editing tracrRNA or ‘petracrRNA’) (Figure 1a, bottom; Figure 1b). Previous reports have shown that the distal double-stranded RNA region of the crRNA:tracrRNA junction (also known as the repeat:anti-repeat junction) is not necessary for Cas9 function; this region is replaced by the GAAA tetraloop sequence in the standard sgRNA constructs (Jinek et al., 2012). Therefore, we replaced this sequence with other complementary RNA sequences and appended an additional RNA pseudoknot structure at the end of both molecules to inhibit early degradation of split RNAs, similar to our testing of sgRNA/petRNA above.

We cloned six pairs of crRNA-petracrRNA pairs, varying sequences that are likely to drive dimerization between crRNA and petracrRNA, but less likely to be essential for interaction between the pegRNA and prime editor (Figure 1c). Using the 10 bp dimerization sequence found in the standard sgRNA, we observe editing efficiency at HEK3 of 15% (Figure 1d), which was 37% of the editing efficiency of the standard epegRNA programming of the same edit (‘eCTT control’) with the same PE4max prime editor. When different dimerization sequences were placed on crRNA and petracrRNA, the editing efficiency varied between 15% and 25%. The editing efficiency was further enhanced to 28% by flipping A-U pairs at the bottom of repeat:anti-repeat duplex, which is 76% of the editing efficiency of the eCTT control. In contrast, removing the dimerization sequence reduced the editing efficiency to 2.7%, which further reduced to 0.3% when the upper part of the Cas9-binding duplex was removed (Figure 1c and d; short-1 and short-2 designs). To verify that the observed editing was dependent on RNA-RNA hybridization, if we paired crRNA and petracrRNA with unmatching dimerization sequences, we consistently observed low editing efficiency (2–3%) (Figure 1e).

In summary, we tested three strategies to split the pegRNA into two molecules (Figure 1a). Among these, splitting the pegRNA at the repeat:anti-repeat portion is the most promising strategy, and can be potentially extended to the general gRNA used with CRISPR-Cas9 (Figure 1a, bottom; Figure 1b–e). The resulting rates of prime editing are strongly dependent on the complementarity between the repeat:anti-repeat region of crRNA and petracrRNA. Furthermore, our design mimics the functional molecules within the native CRISPR-Cas9 system, initially discovered as the dual-RNA-guided system (Jinek et al., 2012). Therefore, we pursued this strategy in further developing a molecular proximity sensor that drives genome editing.

To convert protein-protein interaction events into genome editing events, we reasoned that the RNA dimerization domain within crRNA and tracrRNA could be replaced with an RNA-based protein dimerization domain, such that specific protein-protein interactions or prolonged proximity would aid in the formation of active gRNA that would then bind to Cas9 (Figure 2a). To date, many specific protein-RNA interaction parts have been identified, including the MS2 RNA aptamer that binds to the MCP domain (Johansson et al., 1997). Among different RNA-protein pairs, we chose two pairs, MS2-MCP and BoxB-LambdaN (Katrekar et al., 2022; Legault et al., 1998; Salstrom and Szybalski, 1978), which can be used as adaptors for using protein-protein interactions to drive RNA-RNA interactions.

We designed a crRNA and petracrRNA pair, for which previously identified dimerization sequences of the upper portion of the repeat:anti-repeat duplex were replaced with MS2 and BoxB RNA aptamers. To aid the formation of the proper bulge within the RNA duplex (A and AAG in crRNA and petracrRNA, respectively), we replaced the original sequence of GCUA in crRNA with GCGC, and UAGC in petracrRNA with GCGC. Finally, we added evoPreQ1 RNA pseudoknot sequence on both RNA molecules to reduce RNA degradation from 3’-ends, resulting in crRNA-MS2 and BoxB-petracrRNA.

To test whether crRNA and petracrRNA modified with protein-binding aptamer sequences can be used to drive genome editing, we constructed a single protein where the MCP and LambdaN domains were fused with a flexible linker (MCP-LambdaN), with the intent of bringing crRNA-MS2 and BoxB-petracrRNA into close proximity to form active pegRNA. When we transfected HEK293T cells with plasmids expressing MCP-LambdaN, PE4max (Chen et al., 2021), crRNA-MS2, and BoxB-petracrRNA that target HEK3 locus and program for CTT insertion at +0 position, we observed 15% editing efficiency, which was 52% of the editing rate achieved by transfecting a single, standard epegRNA-expressing plasmid that programs the same edit along with the PE4max-expressing plasmid (‘eCTT control’) (Figure 2b). When the MCP-LambdaN-expression plasmid was substituted with a GFP-expressing filler plasmid (‘Filler GFP plasmid’ condition in Figure 2b), editing efficiency dropped to 6% of the eCTT control, indicating that crRNA-MS2 and BoxB-petracrRNA can promote genome editing to a limited extent, presumably by forming an active pegRNA complex even without the additional dimerization domain.

We next sought to extend this approach to link arbitrary protein-protein interactions to genome editing. For this, we designed protein components that can induce the formation of pegRNA complexes containing crRNA-MS2 and BoxB-petracrRNA. As above, because the editing efficiency can vary depending on the transfection efficiency (30–60% depending on the culturing condition and confluency of the cell culture), we continued to use a ‘normalized editing efficiency’ scale where the observed editing efficiency is scaled to the eCTT control to facilitate comparison across experiments.

We selected three known pairs of interacting protein domains and appended MCP and LambdaN domains with several nuclear localization sequences (Chen et al., 2021): (1) GCN4 epitope sequence (20 AA) and single-chain variable fragment (Colby et al., 2004; Lecerf et al., 2001; Tanenbaum et al., 2014; Wörn and Plückthun, 2001) (scFv; 250 AA) that recognizes GCN4 epitope, (2) ALFA-tag sequence (15 AA) and nanobody specific for ALFA-tag (NbALFA; 135 AA) (Götzke et al., 2019), and (3) split-GFP (Cabantous et al., 2005; Ghosh et al., 2000), where 11 beta-sheets of GFP is split into GFP1-10 (220 AA) and GFP11 (17 AA). For the GCN4 epitope design, we constructed two designs where either a single repeat of the epitope (1xGCN4) was used, or four repeats of the epitope (4xGCN4) were strung together, as often done to increase the efficiency of scFv binding to the tagged protein molecule. For the split-GFP design, we also constructed a version without any nuclear localization sequences, to test whether protein localization also affects editing efficiency. Designed proteins were cloned into protein expression plasmids and transfected into cells along with crRNA/petracrRNA/prime editor expressing plasmids.

First, we observe that both epitope-antibody/nanobody interactions were able to induce strong genome editing efficiencies, close to the levels observed with the MCP-LambdaN fusion protein (Figure 2b). Interestingly, the additional GCN4 epitopes in the 4xGCN4 design did not seem to increase editing efficiency, possibly meaning either that GCN4:scFv binding is saturated and additional GCN4 epitopes do not make a difference in bringing crRNA and petracrRNA into proximity, or that only the scFv binding to one of GCN4 epitope contributes to the active pegRNA formation due to spatial constraints. Second, we observe that split-GFP designs generally have lower editing efficiency, possibly due to weaker interaction between GFP1-10 and GFP11 compared to epitope-antibody/nanobody interactions. Finally, all three protein pairs as well as MCP-LambdaN fusion constructs include nuclear localization sequences to enhance genome editing in the nucleus. We also tested the split-GFP construct without a nuclear localization sequence, which resulted in an even lower editing efficiency, possibly indicating that the nuclear localization propensity of proteins tagged with the MCP or LambdaN could affect the effectiveness of P3 editing.

Lastly, we tested whether this strategy could be adapted to facilitate small molecule-based control of genome editing. In the past 30 years, several chemicals have been identified as critical signaling molecules for promoting protein-protein interactions (Schreiber, 2021). We reasoned that the addition of such chemicals to cell culture could be used to control genome editing of specific targets and editing outcomes. To demonstrate the chemical control of P3 editing, we chose rapamycin-induced dimerization of FKBP and FRB protein domains of the mTOR pathway (Banaszynski et al., 2005; Bierer et al., 1990; Brown et al., 1994) from human proteome, and abscisic acid (ABA)-induced dimerization of pyrabactin resistance domain (PYL) and ABA-insensitive (ABI) domain found in plant (Liang et al., 2011; Figure 2c). We observed a strong increase in the editing efficiency of the FKBP-FRB and PYL-ABI pairs upon the addition of small molecules that induce dimerization (Figure 2d). For example, we observed 3.9-fold higher editing upon the addition of 200 nM rapamycin to the FKBP-FRB pair compared to no rapamycin condition (58% vs 15% normalized editing efficiency), and 2.7-fold higher editing upon the addition of 100 μM ABA to the PYL-ABI pair compared to no ABA condition (36% vs 13% normalized editing efficiency) (Figure 2d).

Overall, these experiments demonstrate that P3 editing can be used to control genome editing via specific interactions between a pair of tagged protein domains. Next, we sought to improve the efficiency and specificity of P3 editing by engineering the crRNA-MS2/BoxB-petracrRNA design. We designed 12 pairs of crRNA-MS2/BoxB-petracrRNA guides, varying the base composition and length of the upper 4 bp region within the Cas9-binding region of repeat:anti-repeat duplex (Figure 2—figure supplement 1). Each RNA design was cloned into an RNA expression vector with flanking U6 promoter and TTTTTTT terminator sequences. We transfected HEK293T cells with the mix of four plasmids expressing each component: crRNA-MS2, BoxB-petracrRNA, PE4max, and either split-GFP (GFP1-10 tagged with MCP and GFP11 tagged with LambdaN; to measure the editing efficiency induced by protein-protein interaction) or standard GFP (to measure the background level of editing without protein-protein interaction).

Across 12 designs, we observe a tradeoff between efficiency (ranging between 2% and 53% of normalized eCTT control) and specificity (background editing level without the addition of tagged protein pair ranging between 0.3% and 35%). The efficiency increases with a higher number of G-C base-pairs and annealing length within the varied region, supporting our hypothesis that crRNA-MS2 and BoxB-petracrRNA can form an active pegRNA complex without the addition of protein-mediated proximity, although at a lower rate. However, as the efficiency of P3 editing decreases, the specificity of P3 editing to protein-protein interaction can increase. For example, in our design that uses GAUA:UAUC pairing, we observe 3.8 ± 1.0% normalized editing efficiency with the addition of tagged split-GFP protein, and only 0.27 ± 0.07% normalized editing efficiency without it, a 14-fold increase driven by specific protein-protein interaction. Our optimization effort suggests that the efficiency and specificity of P3 editing can be tuned using different designs of crRNA-MS2/BoxB-petracrRNA, depending on the experimental goal.

While our demonstrations so far coupled the P3 strategy to prime editing, we reasoned that the same strategy could be adapted to other precision editing methods such as base editing (Figure 3a). To confirm this, we tested three different base editors (CBE2 [Komor et al., 2016], CBE4max [Koblan et al., 2018], and ABE8 [Richter et al., 2020]), which can target the HEK3 locus and make either C-to-T or A-to-G edits (Figure 3b). To facilitate compatibility with base editing, we designed BoxB-tracrRNA that lacks the 3’-extension specific to prime editing and cloned it into an RNA expression vector with a U6 promoter. We transfected HEK293T cells with a mix of plasmids expressing: crRNA-MS2, BoxB-tracrRNA, one of the three base editors, and either the LambdaN-MCP fusion protein that brings crRNA-MS2 and BoxB-tracrRNA into close proximity or GFP to serve as a negative control and measure the base-level editing efficiency without a protein component that aids the formation of gRNA complex.

For all three base editors tested, we observe that the addition of LambdaN-MCP increases the editing efficiency (Figure 3c, left), although the change in the editing was greater for CBE4max (3.5-fold increase) and ABE8 (2.1-fold increase) than CBE2 (1.4-fold increase). The observed differences are possibly related to different endonuclease activity, as CBE2 uses deactivated Cas9 instead of Cas9 nickase as in the case of the prime editor and other base editors. Of note, the observed genome editing efficiencies of the P3 strategy for all three base editors (Figure 3c, left) are comparable to our base editing efficiencies measured with standard sgRNA (Figure 3c, right), despite the fact that the P3 strategy uses a three-component system (using crRNA-MS2, BoxB-petracrRNA, and LambdaN-MCP) instead of one sgRNA.

In both prime editing and base editing demonstrations of the P3 strategy, we used the presence of LambdaN-MCP fusion protein to control the genome editing efficiency, forming a synthetic circuit with the input of protein expression and the output of genome editing. Recently, a similar synthetic circuit was developed using ADAR-based RNA editing, where the input of specific target RNA expression can be used to control an output of cargo protein expression. In this system (termed CellREADR [Qian et al., 2022], RADAR [Kaseniit et al., 2023], or RADARS [Jiang et al., 2023]), an ADAR-guide-RNA is specifically designed to bind the target RNA molecule and promote an RNA base editing event that converts an internal stop codon into a sense codon within the ADAR-guide-RNA, thus initiating cargo protein expression.

To combine the ADAR-based RNA sensor and P3 editing concepts, we programmed the cargo protein downstream of the ADAR-guide-RNA to be a fusion protein (e.g. LambdaN-MCP) that is capable of bringing crRNA and petracrRNA in close proximity (Figure 4a). We based our design on the RADARS strategy, which includes an MS2 RNA structure within the ADAR-guide-RNA to aid the recruitment of MCP-tagged ADAR and suppress translation read-through of an internal stop codon. To avoid complications of using MS2 RNA elements in both ADAR-guide-RNA and crRNA, we switched out the MS2/MCP pairing with the PP7 RNA element that binds to the PCP protein (Chao et al., 2008; Olsthoorn et al., 1995; Wu et al., 2014) domain, and confirmed that this could support P3 editing (Figure 4b). We used an ADAR-guide-RNA design that detects IL6 mRNA expression from the RADARS system (Jiang et al., 2023) because the efficiency and specificity of RNA detection have been previously well characterized in the context of the cargo protein expression of luciferase and GFP.

In combining RADARS with P3 editing, we transfected HEK293T cells with a mix of six plasmids that express: MCP-ddADAR, ADAR-guide-RNA-LambdaN-PCP (also referred to as ogRNA in RADARS), IL6 RNA for target RNA (specifically chosen in RADARS system because it is not natively expressed in HEK293T cells) crRNA-PP7, BoxB-petracrRNA, and PE4max (Figure 4c). We observed the normalized editing efficiency of 11.6 ± 0.7%, which was significantly higher than a condition where the IL-6 RNA-expressing plasmid has been replaced with a filler GFP-expression plasmid (8.2 ± 0.3%; p=0.005, where p-values were obtained using the two-tailed Student’s t-test with Bonferroni correction) (Figure 4d).

While this result suggests that we can control genome editing with specific RNA expression events by combining RADARS and P3 editing, we investigated what contributes to the high background level as well as the low editing efficiency. First, when we replace the plasmid expressing ADAR-guide-RNA-LambdaN-PCP with the ADAR-guide-RNA-GFP (with the cargo protein of GFP instead of LambdaN-PCP), we observe the normalized editing efficiency of 5.5 ± 0.6% (Figure 4d). This suggests that the crRNA-PP7 and BoxB-petracrRNA can induce 5.5% normalized editing efficiency without LambdaN-PCP in the system, characterizing the non-specific editing level of P3 editing, and suggesting that the remaining 2.7% of normalized editing efficiency background is due to non-specific editing of the RADARS system, possibly due to leaky expression of LambdaN-PCP even in the absence of IL6 target RNA. Finally, when we replace 50% of ADAR-guide-RNA-GFP expression plasmid with LambdaN-PCP expression plasmid, we observe the normalized editing efficiency to increase up to 34 ± 1%, suggesting that the inclusion of the RNA-controlled protein expression module might be lowering the overall editing efficiency. Overall, these results suggest that while the P3 editing strategy can be combined with other synthetic circuits to control genome editing with various inputs, the efficiency and specificity of control may degrade over combinations of multiple synthetic modules, and that further optimization is necessary.

All crRNA and petracrRNA constructs were cloned using ligation after restriction (T4 DNA Ligase, New England Biolabs), following the protocol outlined in Anzalone et al., 2019. Single-stranded DNAs (IDT) were annealed to have 4 bp overhangs in both ends of double-stranded DNAs, which is a substrate for T4 DNA ligase. The plasmid backbone (pU6-pegRNA-GG-acceptor, Addgene_132777↗) was digested using BsaI-HFv2, and mixed with annealed double-stranded DNA constructs with 4 bp overhangs. At the end of all crRNA and petracrRNA constructs (Supplementary file 1), we added the evoPreQ1 sequence and poly-T terminator sequence. A small amount (1–2 µL) of T4 ligation reaction mix was added to NEB Stbl cell (C3040) for transformation and grown at 37°C for the plasmid DNA preparation (QIAGEN miniprep). The resulting plasmids were sequence-verified using Sanger sequencing (GENEWIZ).

All protein expression constructs () were cloned using Gibson Assembly (NEB, where double-stranded DNA fragments are either ordered from IDT as gBlocks or PCR-amplified from existing constructs with at least 25 bp overlap in sequence). A small amount (1–2 µL) of Gibson Assembly reaction mix was added to NEB Stbl cell (C3040) for transformation and grown at 30°C or 37°C for the plasmid DNA preparation (QIAGEN miniprep). The resulting plasmids were sequence-verified using Sanger sequencing (GENEWIZ). Supplementary file 1

The HEK293T cell line was purchased from ATCC, and maintained by following the recommended protocol from the vendor. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (GIBCO), supplemented with 10% fetal bovine serum (Rocky Mountain Biologicals) and 1% penicillin-streptomycin (GIBCO). Cells were grown with 5% CO₂ at 37°C. The HEK293T cell lines were authenticated and tested for mycoplasma using ATCC Cell Line Authentication and Mycoplasma PCR tests.

For transient transfection, HEK293T cells were cultured to 70–90% confluency in a 24-well plate. For prime editing with crRNA/petracrRNA and without protein components, 375 ng of PE4max enzyme plasmid (Addgene_174828↗), 62.5 ng of crRNA plasmid, and 62.5 ng of petracrRNA plasmid were mixed, and prepared with a transfection reagent (Lipofectamine 3000) following the recommended protocol from the vendor. For transfection with protein components, 250 ng of PE4max enzyme plasmid, 125 ng of protein (MCP-LambdaN, GFP filler plasmid, or 62.5 ng each of two MCP/LambdaN-tagged protein domains), 62.5 ng of crRNA plasmid, and 62.5 ng of petracrRNA plasmid were used in transfection. The transfection mix composition for the experiment with the ADAR-based RNA sensor is described in Figure 4c. Cells were cultured for 3–4 days after the initial transfection, and their genomic DNA was harvested following cell lysis and protease protocol from Anzalone et al., 2019.

The targeted region from collected genomic DNA was amplified using two-step PCR and sequenced using the Illumina sequencing platform (NextSeq). The first PCR (KAPA Robust polymerase) included 1.5 µL of cell lysate, and 0.04–0.4 µM of forward and reverse primers () in a final reaction volume of 25 µL. We programmed the first PCR to be: (1) 3 min at 95°C, (2) 15 s at 95°C, (3) 10 s at 65°C, (4) 90 s at 72°C, (5) 25–28 cycles of repeating step 2 through 4, and (6) 1 min at 72°C. Primers included sequencing adapters to their 3′-ends, appending them to both termini of PCR products that amplified genomic DNA. After the first PCR step, products were added to the second PCR that appended dual sample indexes and flow cell adapters. The second PCR program was identical to the first PCR program except we ran it for only 5–10 cycles. Products were purified using AMPure and assessed on the TapeStation (Agilent) before being denatured for the sequencing run. Supplementary file 1

Sequencing reads from Illumina NextSeq platforms are first demultiplexed using BCL2fastq software (Illumina). Sequencing libraries were single-end sequenced to cover the DNA Tape from one direction. Editing efficiencies were calculated using pattern-matching software such as Regular Expression (package REGEX) in Python, counting correct amplicon reads with or without intended edits. For prime editing, CTT insertions to the HEK3 locus (Anzalone et al., 2019) were counted, and editing efficiencies were calculated by: reads with CTT insertions/all reads matching the HEK3 locus. In quantifying base editing efficiencies, we first used CRISPResso to identify the top 10 base editing outcomes of the HEK3 locus, and used these editing patterns to calculate editing efficiency as follows: all reads matching the top 10 base editing outcomes/all reads matching HEK3 locus.