Discovery of CRISPR systems in bacteria and archaea has not only drastically increased the spectrum of organisms that we can genetically modify but it has also equipped us with a simple tool for the introduction of a variety of different types of modifications into the genome (Cong et al., 2013; Mali et al., 2013; Wang et al., 2013). Many of the CRISPR-based approaches rely on generating double-stranded DNA breaks that are usually accompanied by genome-wide off-target editing (Tsai et al., 2015; Kim et al., 2015; Kleinstiver et al., 2016b; Tsai et al., 2017). Recently, more precise CRISPR tools—base (Komor et al., 2016; Gaudelli et al., 2017) and prime (Anzalone et al., 2019) editors—have been developed, and these enzymes can introduce modifications into the DNA with a base pair resolution without the requirement of donor DNA templates or the introduction of double-stranded DNA breaks. While current base editor (BE) variants provide efficient editing, they are restricted to certain substitution mutations (Komor et al., 2016; Gaudelli et al., 2017; Kurt et al., 2021; Zhao et al., 2021).

In contrast, prime editors (PEs) can introduce all types of substitutions and/or precise insertions/deletions (indels), however, their efficiency lags behind that of BEs. Prime editor 2 (PE2) consists of a nickase version of Streptococcus pyogenes Cas9 (SpCas9) fused to a reverse transcriptase enzyme (Anzalone et al., 2019). The fused reverse transcriptase can extend the nicked DNA strand using an RNA template that is located on the 3′ terminus of an extended single-guide RNA (sgRNA), called the prime editing sgRNA (pegRNA). By careful design of the pegRNA, modifications can be introduced downstream of the nick generated on the non-targeted DNA strand. The 3′ end of the pegRNA, that is complementary to the non-targeted DNA strand, consists of the reverse transcriptase template (RT) and the primer binding site (PBS). The RT, which contains the mutation(s) to be introduced, also comprises the protospacer adjacent motif (PAM). The PBS is complementary to the 3′ end of the nicked non-targeted DNA strand, with which it forms a hybrid DNA-RNA helix. The optimal length of the PBS and the RT varies from target to target and requires extensive optimization for each different target. The efficiency of prime editing can be increased by nicking the non-edited strand (prime editor 3—PE3) at the expense of potentially generating unwanted indels at the targeted locus (Anzalone et al., 2019). The effect of the nicking varies depending on its position and it requires further optimization. In some cases, the efficiency of prime editing can be further increased by also altering the PAM sequence with the desired mutation this may prevent the PE from nicking the sequence again. This method can reduce the chance of introducing unwanted indels alongside the intended mutations, thus increasing the occurrence of precise modifications (Anzalone et al., 2019).

The development of improved PE variants can be forecasted in view of the history of BEs which are now available with highly improved features and efficiency (Kim et al., 2017; Komor et al., 2017; Koblan et al., 2018; Huang et al., 2019; Gehrke et al., 2018; Zafra et al., 2018; Gaudelli et al., 2020). The aim of our study was to develop a reporter system for an easy, fluorescence-based detection of prime editing outcomes that allows maximum flexibility for the target sequences and pegRNA designs to be tested on it. Several systems have been developed for reporting base editing that use BEs for the generation or alteration of a start or stop codon (Katti et al., 2020; Wang et al., 2020), or to rescue a disruptive amino acid and subsequently recover functions of antibiotic resistance genes or fluorescence proteins (Martin et al., 2019). Alternatively, a non-synonymous mutation in the chromophore of a fluorescent protein that induces fluorescence spectral change has also been explored as a method to monitor base editing activity (Coelho et al., 2018; Standage-Beier et al., 2019). Reporter systems have also been demonstrated for prime editing, but they were restricted to very few target sequences (Lin et al., 2020; Sürün et al., 2020) and/or showed a rather low signal (Katti et al., 2020).

We also wanted to find out whether prime edited mammalian cells could be identified and enriched by using a plasmid-based surrogate marker for chromosomal DNA modifications, as demonstrated before with Cas nucleases (Ramakrishna et al., 2014; Grav et al., 2015) and BEs (Katti et al., 2020; Coelho et al., 2018; St Martin et al., 2018; Tálas et al., 2021).

We aimed to develop a reporter system that possesses several key features. It should be a transient plasmid-based system that is not restricted to one or a few cell lines nor does it require extensive work, such as the generation of cell lines. It needs to be based on a gain-of-function fluorescent signal with minimal background so it can be detected in the timeframe of a transient system. The sequence requirements of efficient prime editing are not yet fully understood (Anzalone et al., 2019; Kim et al., 2021); thus, for the widespread application of a reporter system, it is crucial that the sequence of the target and the flanking nucleotides of the position to be edited can be freely interchanged. We have recently developed a base editor activity reporter (BEAR), which meets these criteria (Tálas et al., 2021), and it has the potential to be converted into a tool suitable for reporting on prime editing activity. BEAR is based on a split GFP protein separated by the last intron of the mouse Vim gene. The sequence of the functional splice donor site (or 5′ splice site) is altered in such way, that splicing and therefore the GFP fluorescence is disrupted, however, they can be restored by applying adenine or cytosine BEs (Figure 1—figure supplement 1A). Sequence alterations in the intronic sequence are well tolerated, which gave us the flexibility to investigate numerous target sequences. Using the information acquired during the development of the BEAR system (Tálas et al., 2021), we have designed a plasmid that contains an inactive splice site, which can be activated by the action of a PE harboring an appropriately designed pegRNA (Figure 1A). Cells containing plasmids with activated splice site sequences will then be able to efficiently express GFP, which can be quantified by flow cytometry. Therefore, we name assays that exploit this design as prime editor activity reporters (PEARs).

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There are differences between the features of BEAR and PEAR. The editing windows of BEs exploited for the BEAR system are located PAM distal, 5′ of the nick (Figure 1—figure supplement 1B). PEs on the other hand can introduce mutations 3′ of the nick, a region that contains the PAM sequence (Figure 1A, Figure 1—figure supplement 1C). To ensure maximum flexibility for the PE target, we kept the target sequence in the intron by placing the PAM to the complementary strand. As a result, the target sequence can be freely adjusted in its entire length (Figure 1A). Thus, while BEAR could be used with a few million of different target sequences, PEAR offers unrestricted sequence variations for the entire SpCas9 target. On the contrary, PEAR is similar to BEAR in that it is permissive with respect to the interchangeability of the targeted nucleotides (here the splice site and the splice site flanking nucleotides; Tálas et al., 2021). No other fluorescence-based reporter systems have demonstrated such degree of flexibility before (Katti et al., 2020; Lin et al., 2020; Sürün et al., 2020).

To identify the optimal PBS, RT, and complementary nick combination, HEK293T cells were transfected with the target plasmid (named PEAR-GFP) carrying the inactive splice site sequence to be edited, and several combinations of plasmids coding various pegRNAs, sgRNAs (targeting the PEAR-GFP plasmid), and PE2 protein. The number of GFP-positive cells was then measured by flow cytometry (Figure 1—figure supplement 1D). The heatmap in Figure 1B shows that the PEAR-GFP plasmid can be efficiently edited by many of these combinations of PBS, RT, and nicking position. The most efficient editing occurred, when a 10-nucleotide long PBS (PBS-10) and a 24-nucleotide long RT region (RT-24) were applied. Figure 1B also shows that in line with expectations, editing efficiency is generally higher when the complementary strand is nicked, than when it is not (Anzalone et al., 2019). The most effective complementary strand nicking site was position +17 in the case of most PBS and RT length combinations. In all conditions, PBS-10 gave greater values than PBS-13 and PBS-16. Comparing the RTs, the efficiency of the editing was in the order of RT-24>RT-33>RT-16 in most conditions (16 out of 18). These results are consistent with the concept that the effect of the length of PBS and RT on editing efficiency is primarily independent of one another (Kim et al., 2020). It is also in line with the current optimization practice, where first, the length of the PBS is optimized using a given RT length, and then the length of the RT is optimized using the PBS selected in the first step. Thus, these experiments support the hypothesis, that PE efficiency is governed by the same factors when using a PEAR plasmid, as demonstrated earlier on chromosomal targets (Anzalone et al., 2019; Kim et al., 2021).

Before proceeding to the further characterization of the sequence dependence of PEAR, we examined whether the fluorescent signal originates exclusively from the recovered splice site sequence. PEs themselves generate various amounts of indels that may lead to the incidental restoration of fluorescence. In this experiment, to report on prime editing, we used a previously constructed plasmid (Tálas et al., 2021) that expresses a split mScarlet (BEAR-mScarlet). We wanted to simultaneously detect caused indels in a cell line by the means of a disruption assay, as well as prime editing on the BEAR-mScarlet plasmid. We transfected the BEAR-mScarlet plasmid along with either an SpCas9 nuclease, a nickase or a PE protein-coding plasmid into HEK293.EGFP cells (harboring the coding sequence of EGFP integrated into the genome). We also transfected a pegRNA to correct the splice site on the BEAR-mScarlet plasmid along with sgRNA(s) targeting the GFP coding sequence. This design allowed us to co-monitor indel generation via GFP disruption and prime editing via restoring mScarlet fluorescence on the BEAR-mScarlet plasmid. As expected, while all three variants—SpCas9 nuclease, nickase, or PE—demonstrated GFP disruption activity, only PE restored mScarlet fluorescence, indicating that PEAR specifically reports on prime editing activity and is insensitive to the potential presence of indel background (Figure 1—figure supplement 1E).

Using the optimal PBS-RT combination found in Figure 1 constructs with different inactive splice site sequences were edited by PE2 to the same active splice site sequence (as in Figure 1B) by either deleting or inserting nucleotides in six and seven cases, respectively (Figure 1C). Interestingly, there seems to be no connection between the efficiency and the complexity of the editing. Figure 1C demonstrates that all types of sequence alterations can be examined using the PEAR system.

The versatility of the PEAR assay, with respect to sequence modifications, lies not only in the fact that an active splice site can be restored from seemingly any inactive sequence as long as the change can be efficiently implemented by prime editing, but also that the active splice site sequence itself can be varied. To demonstrate this, we corrected three additional active splice sites from different inactive sequences (Figure 1D).

In order to verify that the same factors influence prime editing on a plasmid as on genomic targets, we compared prime editing efficiency on previously constructed plasmids and two HEK293T cell lines (HEK-BEAR-GFP and HEK-BEAR-mScarlet—Figure 2—figure supplement 1; Tálas et al., 2021) that contain a chromosomal copy of these plasmids harboring an interrupted GFP or mScarlet sequence, respectively. As the plasmids (BEAR-GFP and BEAR-mScarlet) contain the same sequence around the inactive splice site as in the cell lines, they can be compared pairwise. The sequences to be edited in the two cell lines were designed to be used with base editing that requires a PAM sequence 5′ upstream of the edit. By contrast, prime editing requires a PAM position 3′ downstream. Thus, we needed to identify another PAM site that would allow the application of prime editing on these sequences. For BEAR-GFP, there is just one PAM, but for BEAR-mScarlet there are two PAMs in the primary editing window, and this gave the opportunity to perform the comparison for two targets for BEAR-mScarlet.

In case of the GFP expressing cells, we tested 36 different conditions for both the cell line and the BEAR-GFP plasmid: all possible PBS-RT-second nick site combinations. The heatmap in Figure 2A shows that PBS-13 and RT-22 or RT-26 in combination with the second nick site at position +103 results in maximum efficiency for the BEAR-GFP plasmid. In the absence of a second nick, the efficiency of editing was considerably lower. The most ideal conditions for prime editing were the same both in the plasmid and in the cell line (Figure 2A). The overall pattern of the two heatmaps is indisputably similar, exhibiting strong correlation (r=0.89, Figure 2—figure supplement 2A). In case of the cell line, a somewhat lower general editing efficiency is apparent compared to that with the plasmid. This difference is likely due to the much higher copy number of the plasmids present in the cell.

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In case of the mScarlet expressing cells, we compared the efficiency of prime editing with both target 1 (Figure 2B) and target 2 (Figure 2—figure supplement 2B), by exploring 36 different combinations of PBS, RT, and second nick site for each target. Target 1 gave higher prime editing efficiencies, with the top working PBS being 10 nucleotides long in the case of both the BEAR-mScarlet plasmid and the cell line (Figure 2B). The two heatmaps exhibit a remarkably similar pattern, and there is a strong correlation of the editing efficiency in the cell line and the BEAR plasmid (r=0.93; Figure 2—figure supplement 2C). Similar outcomes can be seen with target 2 where the best PBS length is also 10 nucleotides long for both the BEAR-mScarlet plasmid and the cell line (Figure 2—figure supplement 2B). Comparing plasmid and genomic results also revealed a remarkably similar pattern (r=0.92; Figure 2—figure supplement 2D). With the same intron being in all three plasmids, we used the same three sgRNAs for secondary nicking in combination with all pegRNAs and plasmids or cell lines. For all three targets studied, regardless of whether in plasmids or in cell lines, the three secondary nick positions affected the effectiveness of prime editing in the same way. Collectively, these results strongly imply that the main features of prime editing and factors affecting its efficiency are reflected accurately within our system.

Post-transfection selection of edited cells by fluorescence-activated cell sorting (FACS) or antibiotics have proven to be effective methods for the enrichment of genetically modified cells within a population, given that the SpCas9 coding plasmid co-expressed a protein that is fluorescent or bears antibiotic resistance (Katti et al., 2020; Standage-Beier et al., 2019; Ramakrishna et al., 2014; Grav et al., 2015; St Martin et al., 2018; Tálas et al., 2021). Experiments have also shown that selecting for cells with a surrogate marker, that is subjected to the same type of genetic modification (knockout, HDR, or base editing) as the intended edit, results in higher enrichment of successfully edited cells, than when solely selecting for transfection markers (Katti et al., 2020; Coelho et al., 2018; Ramakrishna et al., 2014; Grav et al., 2015; St Martin et al., 2018; Tálas et al., 2021; Yan et al., 2020) suggesting that some individual cells allow for higher editing efficiency. This phenomenon could be due to the nuclease being able to exhibit higher activity, or the activity of DNA repair systems could be considerably more engaged in these cells. We proposed that the level of enrichment, that can be achieved using the transfection marker alone, can be exceeded by enriching cells in which a marker protein in a plasmid is simultaneously prime edited. The PEAR-GFP plasmid was used in these experiments as a surrogate marker with previously optimized RT and PBS conditions (Figure 1B).

To test this principle, first, we transfected the BEAR-mScarlet cell line edited in Figure 2 with the PEAR-GFP plasmid along with two pegRNAs (also coding BFP as a transfection marker) one targeting the cell line and one targeting the plasmid to explore if prime editing on the plasmid is associated with genomic (mScarlet) editing. We measured the ratio of the mScarlet positive cells in (1) all measured cells, (2) within cells that either express BFP for transfection marker enrichment, or (3) express GFP (regardless of BFP expression) for PEAR enrichment. For this purpose, we chose two efficient PEAR-GFP targeting pegRNAs tested earlier (Figure 1B). Figure 3A shows that transfection marker enrichments significantly increased the ratio of edited and non-edited cells: gating for the BFP-expressing cells increased the percentage of the edited population from 18% to 24% with both pegRNAs tested. PEAR-enrichment resulted in a considerable increase compared to the transfection enrichments in case of both pegRNAs (57% and 76% mScarlet expressing cells, respectively). These results demonstrate that prime edited cells can be substantially enriched when using a PEAR plasmid as a surrogate marker.

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To test the applicability of PEAR for enriching the prime edited population in endogenous targets generating single-nucleotide substitutions, we chose five genomic targets (EMX1, RNF2, FANCF, HEK3, and HEK4) which have been investigated previously (Anzalone et al., 2019). Here, BFP was used as the indicator of transfection efficiency, and we constructed a new plasmid (PEAR-GFP-2in1) as a marker for successful prime editing, which contains the pegRNA 1 (the best pegRNA in Figure 1B) expression cassette on the same plasmid. We co-edited the genomic targets and the PEAR-GFP-2in1 plasmid, and three days after transfection, cells were sorted into three fractions: (1) all single living cells, (2) BFP-positive cells, and (3) GFP-positive cells (Figure 3B). From the sorted cell populations, editing was quantified by NGS. Since HEK293T cells are very susceptible to prime editing due to the fact that their mismatch repair system is partially defective (Chen et al., 2021; Trojan et al., 2002), we also attempted enrichment in K562 and U2OS cells, and on a human embryonic stem cell line HUES9, that are all less susceptible to prime editing (Figure 3C–E).

The editing rates increased using transfection marker enrichment by up to 1.65-fold; however, they all showed a significantly greater increase when the PEAR enrichment was used. PEAR enrichments ranged between about twofold and fourfold, reaching up to 62% and 76% editing at FANCF site in K562 and HEK293T cells, respectively. Editing the same target in HUES9 cells showed a 7.8-fold increase with 28% PEAR-enriched editing, whilst editing without enrichment remained below 4% in HUES9 cells (Figure 3C and Figure 3—source data 1).

We also analyzed whether PEAR-enrichment alters the specificity (defined here as editing%/indel%) of editing, by quantifying the generated indels by NGS from the same samples (Figure 3D). Enrichments increased the incidence of indels at the edited sites in the population in 8 out of the 13 edits. However, the extent of these increments is generally low: the specificity slightly decreased for only one edit, while it slightly increased in five cases (Figure 3E).

Furthermore, we determined off-target prime editing from enriched samples at previously reported (Tsai et al., 2015; Anzalone et al., 2019; Kleinstiver et al., 2016a) SpCas9 off-target sites for four targets in HEK293T cells. Only 1 of the 15 off-target sites showed detectable editing (Figure 3F) and indel formation (Figure 3G) that was also enriched by PEAR enrichments. For the remaining 14 sites, PEAR-enrichment did not increase either off-target editing or indel formation to a detectable level. Thus, these data suggest that PEAR enrichments substantially increase prime editing in various cell lines without compromising neither on-target nor off-target specificity.

The PEAR plasmid may also contribute to undesired DNA modifications during editing. Figure 3—figure supplement 1 shows that integration of the active plasmid to the genome is minimal; however, since plasmids tend to integrate into the genome, albeit with low efficiency, unedited PEAR plasmids are likely to still integrate into the genome. Clones should be checked for the absence of the transfected plasmids, such as the PE-expressing and the PEAR plasmid. We have developed PCR primers (Supplementary file 1) for detecting whether the PEAR plasmid has been integrated into the genome.

It is also important, that the pegRNA used for co-editing the PEAR plasmid has no on-target site in the genome and has only minimal genome-wide off-target effect in order to decrease the potential off-target events during PEAR-enrichments. However, to check the genome-wide off-targets of SpCas9 with such sgRNA is inherently a challenging task, since, due to the lack of a reference on-target site in the genome, it is difficult to interpret the results of a GUIDE-seq experiment aiming to determine its off-targets. To overcome this problem, we chose two EGFP targets (sites 2 and 7) that have no on-target sites in the genome but can be tested for off-targets in HEK293.EGFP cells. This cell line provides the reference on-target sites that are normally not present in the genome of human cells. The genome-wide off-target sites of WT-SpCas9 nuclease for EGFP site 2 have been determined previously (Kulcsár et al., 2020) and for EGFP site 7, they were determined in this work by GUIDE-seq. Both sgRNAs proved to have no detectable off-targets (Figure 4A).

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For further experiments, we constructed PEAR plasmids using these targets that can be edited without off-targets. As the GFP targets are also present in the original PEAR-GFP plasmid, by introducing silent mutations we created six mismatches in the case of EGFP site 2 and altered the PAM sequence in the case of EGFP site 7 to make sure that the pegRNA will not target the GFP coding sequence on the plasmid. To find an effective pegRNA to edit the new PEAR plasmids, three PBS lengths were tested for each spacer sequence with the 24-nucleotide long RT sequence identified in Figure 1B as the most efficient (Figure 4B). The highest editing was found with EGFP site 2 with a 12-nucleotide long PBS sequence. This plasmid—named PEAR-GFP-2in1-2.0—was used in subsequent experiments to enrich additional nine target sites installing three insertions, deletions, and substitutions each (Figure 4C–E). These results proved that the PEAR-GFP-2in1-2.0 plasmid is also suitable for the enrichment of prime editing and confirmed the efficiency of PEAR-enrichment for various types of editing. The editing rates by PEAR-enrichments increased from 2.1- to 4.6-fold and up to 84% (Figure 4C). Figure 4D and E show that the increase in editing rates is not accompanied with a decrease in specificity in the case of any of the nine target sites.

Prime editing provides a great flexibility for genomic modifications without having to supply a donor DNA or needing a double-stranded DNA break (Anzalone et al., 2019). This technology could revolutionize the rapidly expanding universe of genome editing. Prime editing technology has readily been translated from mammalian models (Anzalone et al., 2019; Sürün et al., 2020; Kim et al., 2020; Liu et al., 2020; Schene et al., 2020) to plants (Lin et al., 2020; Jiang et al., 2020; Lu et al., 2021; Xu et al., 2020) and Drosophila (Bosch et al., 2020). Despite its great potential, during the preparation of our manuscript, we have found surprisingly few studies that had demonstrated the practical use of prime editing in mammalian cells (Sürün et al., 2020; Schene et al., 2020), which is a vast underestimation of the true potential flexibility of this method. This might be due to the extensive optimization it requires and the relatively low editing efficiency that can be achieved at a subset of targets. In this aspect, any approach that can substantially increase the editing efficiency is critical to aid its generalized use. Here, we developed PEAR, a sensitive fluorescent assay for the monitoring of prime editing activity in mammalian cells, and we also showed that prime editing occurs at much higher frequencies in those individual cells, in which the PEAR plasmid is co-edited. In fact, enrichment with a transfection marker resulted in only a 1.45-fold increment on average in all experiments, whilst PEAR enrichments tripled the number of edited cells (Figures 3A, C ,, 4C). Furthermore, we constructed a PEAR plasmid (PEAR-GFP-2in1-2.0) which bears a pegRNA that has no detectable off-target sites in the human genome as assessed by GUIDE-seq. This plasmid was used to successfully enrich additional targets with the same efficiency. Thus, the application of PEAR as a surrogate marker, as demonstrated in four cell lines including HUES9 cells, could substantially contribute to the more widespread use of prime editing by considerably increasing the number of potential targets, which may now be edited with appropriate efficiency.

PEAR is also a versatile tool that can be utilized for the characterization of different PEs. We examined more than 250 conditions combining different sgRNAs, PBS- and RT-lengths, and nick sites, and our results confirmed that the efficiency of prime editing to modify PEAR plasmids is governed by the same factors as prime editing in genomic context (Figure 2 and Figure 2—figure supplement 2). The tolerance of the splice donor site for substitutions (Figure 1D; Tálas et al., 2021) makes the target region sequence of the PEAR plasmid readily adjustable. This versatility allows the user to examine the effect of sequence features and other factors affecting the efficiency of prime editing in a systematic manner that cannot be explored otherwise using genomic targets. Our approach, beyond its general use, may also assist the development of more efficient PEs or variants with enhanced characteristics in the future.

Owning to the relatively low efficiency of prime editing with many potential target sequences, PEAR is expected to become a widespread tool to be used in a wide variety of biomedical and biotechnological applications. PEAR may be an especially useful tool to be used with hard-to-edit cells or where the efficiency of PEs is particularly compromised.

The following plasmids developed in this study are available from Addgene: pDAS12125_PEAR-GFP (#177178), pDAS12124_PEAR-GFP-preedited (#177179), pDAS12069-U6-pegRNA-mCherry (#177180), pDAS12222-U6-pegRNA-BFP (#177181), pDAS12230_pegRNA-PEAR-GFP(10PBS-24RT)-mCherry (#177182), pDAS12137_sgRNA-PEAR-GFP-nick(+17)-mCherry (#177183). pDAS12342_PEAR-GFP_2in1 (#177184), pDAS12395_PEAR-GFP-2.0-preedited (#177185), and pDAS12489_PEAR-GFP-2in1-2.0 (#177186). GUIDE-seq and NGS sequencing data are deposited at NCBI Sequence Read Archive: PRJNA779199.

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Decision letter after peer review:

Thank you for sending your article entitled "PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells" for peer review at eLife. Your article is being evaluated by 4 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by Didier Stainier as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Jennifer Hamilton (Reviewer #3).

The main concern expressed by all four reviewers is that the PEAR technology has so far limited usefulness, at least as presented in the current version of the manuscript. In order to consider this manuscript for publication, we would like to see additional experiments demonstrating that this technology can be used to detect and enrich for PE edits in difficult-to-edit cell types and more elaborate prime edits beyond single nucleotide substitutions, such as insertions/deletions.

Reviewer #1:

Current reporter systems to detect prime editing outcomes are restricted to few target sequences and/or show a low signal. The aim of the study was to develop a reporter system for an easy, fluorescence-based detection of prime editing outcomes, that allows maximum flexibility for testing the target sequences and pegRNA designs in various cell types. Moreover, the authors aimed to use the developed reporter system as a plasmid-based surrogate marker to select/enrich for cells that undergo PE mediated chromosomal DNA modifications.

Strengths

The reporter is based on a split GFP protein separated by the last intron of the mouse Vim gene. The sequence of the functional 5' splice site is altered in such way, that splicing and therefore the GFP fluorescence is disrupted, however, they can be restored by prime editor harboring an appropriately designed pegRNA. In this case the DNA sequence that base pairs to the spacer region of the pegRNA is downstream of the splice site. This setup provides a gain-of-function fluorescent signal with minimal background and an unlimited flexibility to test and design pegRNAs for multiple target sequences and surrounding sequence context.

Since this setup allows to replicate the chromosomal site of interest on the surrogate reporter plasmid, it enables enrichment (~3 fold) of the cells that get edited at the chromosomal DNA site.

Since the reporter is on plasmid, it is easy to use, no need to create cell lines. In principle, it is not restricted to specific cell lines.

Weaknesses

Although the paper does have strengths, the main weakness of the paper is that not all of these strengths are directly demonstrated. In particular:

The authors imply that the surrogate reporter system they've developed is not restricted to one or a few cell lines. However, all the experiments were performed in one cell line (HEK293T).

The advantage of using prime editing instead of more efficient base editing is that this approach allows the introduction of all types of substitutions and/or precise indels. The authors demonstrated that their reporter system works well when PE is designed to substitute several nucleotides but there were no experiments performed to demonstrate how well this reporter system performs when designing pegRNAs to perform small or large indels.

1. The authors imply that the surrogate reporter system they've developed is not restricted to one or a few cell lines. The authors should demonstrate this by performing prime editing in different cells in the presence of their surrogate reporter system. It would be interesting to see if the PEAR would allow significant enrichment of PE edited cells such as K562 or U2Os which have quite low prime editing efficiencies (PMID: 31634902).

2. The authors aimed to demonstrate that indels introduced by nCas9 do not turn on GFP fluorescence (Figure 1c). However, this could result due to nCas9 being inactive during this experiment. The authors should perform an additional control experiment showing that nCas9 was active and introduced indels but at the same time did not turn on GFP fluorescence.

3. The advantage of using prime editing instead of more efficient base editing is that this approach allows the introduction of all types of substitutions and/or precise indels. The authors demonstrated that their reporter system works well when PE is designed to substitute several nucleotides. Is it possible to use this system for designing pegRNAs to perform small or large indels?

Reviewer #2:

Simon and colleagues report on the development of a fluorescent reporter system, PEAR, which facilitates the enrichment and isolation of cell populations edited by prime editors (PEs). The strength of this reported method is that it can increase the likelihood of identifying cells that have undergone successful prime editing at target loci. In addition, the relative ease of use of the reporter system will make it easily adoptable by other researchers. However, there are several weaknesses in the manuscript in its current form including but not limited to the following: (i) Relative limited number of target loci that were used to validate the PEAR-based system, (ii) Lack of demonstration that the PEAR-based system works across multiple cell lines including those recalcitrant to genome modification, (iii) Missing off-target analysis as it relates to PEAR-induced editing events, (iv) Insufficient statistical analysis, and (v) Missing comparisons to gold-standard methods of editing enrichment. Because of these significant weaknesses, I do not recommend the manuscript to be published in its current form.

I recommend the authors address the following points prior to publication:

(1) As it relates Figure 3b, the standard for analyzing these types of editing events would be an NGS/HTS analysis of the targeted loci to see the individual allelic outcomes from the editing. As in currently stands, the authors only perform Sanger sequencing of the bulk populations. On a related note, does PEAR-based enrichment also increase the frequency of indel formation relative to transfection or no-enrichment? The current Sanger sequencing-based analysis does not provide this sort of insight.

(2) In Figure 3b, the authors do not perform any off-target analysis of prime editor activity. At minimum, the authors should PCR amplify the most-likely off-target loci and confirm that PEAR-based enrichment does not increase likelihood of off-target events. In addition, the authors should ensure that the pegRNA and nicking sgRNA targeting the PEAR plasmids do not induce off-target edits.

(3) In Figure 3b, the authors perform comparison against reporters of transfection. However, much of the field are using reporters of expression for enriching gene editing events. The authors should make similar comparisons to demonstrate the utility of their method.

(4) The authors only perform analysis of PEAR in bulk sorted cell populations. Although such enrichment is useful the authors do not provide any analysis of clonally isolated cell populations to determine the utility of PEAR in such applications.

(5) The authors only perform analysis on 3 genomic loci which does not provide a good indication of the broad utility of PEAR-based enrichment strategies. It is recommended that the authors demonstrate the utility of PEAR-based enrichment methods in the context of additional loci including those that are recalcitrant to typical PE strategies. In addition, the authors should demonstrate the utility of PEAR in the context of other types of PE-driven edits (i.e. small deletions, insertions) in addition to single base pair changes.

(6) Is the fluorescent signal associated with the PEAR plasmids transient? Are there a certain number of passages required to lose the fluorescent signal? Along similar lines, the authors should at least discuss what strategies can be taken to ensure there is not genomic integration of the PEAR reporter plasmids.

(7) The authors only perform PEAR based enrichment with HEK293 cells, which are very amenable to gene editing. To demonstrate the broad utility of this tool the authors should perform additional experiments in other cells lines, including those such a primary cells or pluripotent stem cells which are resistant to genetic modification.

(8) Throughout the manuscript, information about statistical analysis, number of biological replicates, and other information related to scientific rigor are missing.

Reviewer #3:

Prime editing is an exciting new tool in the CRISPR genome editing toolbox which can introduce targeted sequence insertions, deletions and base substitutions. New strategies to enrich for prime edited cells would abrogate the need for time-consuming single-cell sorting and clonal expansion. Simon and colleagues sought to develop a method for easily recovering prime edited cells. Such a method would be useful, as prime editing thus far has only reached moderate levels of efficiency. To achieve this, the authors developed a fluorescence-on, plasmid-based reporter that, when successfully prime edited, repairs a damaged splice site and leads to expression of a fluorescent transgene.

The authors nicely demonstrate that sorting out fluorescent, reporter-positive cells enriches for successful prime editing in the cellular genome. Prime editing of the surrogate plasmid reporter robustly enriched for successful prime editing in a genomically-integrated transgene. Interestingly, this method was also successful when one pegRNA drove prime editing of the plasmid reporter and one pegRNA directed a single base change in endogenous genes (FANCF, RNF2, HEK3).

One key feature of prime editing is that it can be used to make complex genomic alterations such as templated sequence insertions. This has been used to epitope tag endogenous genes (FLAG/His6 tags) and make prime editing distinct from base editors, which aim to alter single base pairs. A weakness of the work presented by Simon and colleagues is that they limit testing of the PEAR reporter to enrichment of cells following single base pair modifications in endogenous genes. In this context, the PEAR reporter was very good at enriching for prime edited cells but it is an open question as to how well PEAR would allow for the enrichment of more complex prime edits (such as multiple base changes or sequence insertions/deletions). Additionally, it is unclear how well the PEAR reporter would enrich for prime-edited cells edited at low efficiency (<10%), where enrichment would be most valuable. Lastly, the PEAR reporter functioned well in the cell line tested (HEK293Ts) but it is unknown how this strategy would work in other immortalized cell lines or primary cells. The PEAR reporter would be a boon if it could successfully enrich for prime edited cell types that are not amenable to single cell sorting and clonal expansion (such as primary cells or cell lines that are difficult to grow up from a single cell, such as Calu-3s).

Together, the authors achieved their aim of developing a fluorescence-on reporter that allows for the enrichment of prime edited cells, both when the prime edit is made in a genomically-integrated transgene or at an endogenous genomic site. This tool will be useful for the genome editing community and/or researchers who wish to introduce minimal base pair changes in HEK293T cells.

In the discussion, the authors state that "The tolerance of the 5' splice site for substitutions makes the sequences of the target region of the PEAR plasmid easily adjustable." The manuscript would be strengthened if the authors demonstrate that this is true.

Reviewer #4:

The manuscript by Dorottya Simon and colleagues describes a reporter of prime editing activity, dubbed PEAR. Prime editing is a next-generation CRISPR-based genome editing strategy. Prime editing relies on a CRISPR enzyme appended with a reverse transcriptase and a stretch of single-stranded RNA that can be used as a template for a reaction that extends a genomic DNA "primer", ultimately incorporating the RNA-templated information into the genome. Some incarnations of prime editing incorporate a second, nicking CRISPR enzyme that can increase the frequency of desired outcomes. Prime editing efficiencies typically surpass those of homology-directed repair, but often lag behind those of base editing or cutting-based editing (the introduction of insertions/deletions). Prime editing outcomes remain somewhat unpredictable and the approach would greatly benefit from an improved "guidebook" that outlines the best practices for the technique. To this end, the PEAR reporter may facilitate high-throughput examination of new prime editing strategies, in turn resulting in a greater understanding of the technique.

A strength of this system is the substantial flexibility with respect to the spacer (targeting RNA / targeted DNA) sequence that can be used. A PAM (protospacer-adjacent motif) is required at the "business end" of the prime editing, but otherwise there is considerable freedom in terms of the guide RNA (spacer) that can be used. The PEAR system also allows enrichment of prime-edited populations, allowing the researcher to triple

A potential disadvantage is that the PEAR cassette – whether in plasmid form or following transfection or incorporation – will not be likely to capture all the qualities of a truly endogenous locus. Indeed, these qualities (and their diversity) play a critical role in determining why certain prime editing attempts fare drastically better than others. Loss of this diversity might limit the reporter's capacity for elucidating the determinants of efficient prime editing.

The following comments are intended to improve the manuscript.

"more precise CRISPR tools; base" … this should not be a semicolon. Consider a pair of dashes, and adjusting "developed, that can" to "developed, and these enzymes can"

"BEs'." no apostrophe needed; "of BEs" conveys the possessive.

Figure 1e: the black text can be hard to see when it is in front of the black data points.

"former observations that indels cannot, only substitution mutations" … This seems incorrect.

Reviewer #1:

[…]

1. The authors imply that the surrogate reporter system they've developed is not restricted to one or a few cell lines. The authors should demonstrate this by performing prime editing in different cells in the presence of their surrogate reporter system. It would be interesting to see if the PEAR would allow significant enrichment of PE edited cells such as K562 or U2Os which have quite low prime editing efficiencies (PMID: 31634902).

To facilitate enrichment experiments, we constructed a new PEAR-GFP plasmid which also bears its targeting pegRNA on the same plasmid. We used this plasmid (called PEAR-GFP-2in1) to perform PEAR enrichments on K562, U2OS cell lines and on a human embryonic stem cell line HUES9 on four, three and one genomic target, respectively. We have detected prime editing efficiencies and also indels from the same samples with NGS. The experiments resulted in 1.66-4.16 fold enrichment on K562, in 1.49-2.71 fold enrichment on U2OS and in 7.86 fold enrichment on the HUES9 cell lines compared to the control without enrichment. The results are discussed in the revised manuscript on pages 13-14, lines 308-334 and presented in the revised Figure 3C-E.

2. The authors aimed to demonstrate that indels introduced by nCas9 do not turn on GFP fluorescence (Figure 1c). However, this could result due to nCas9 being inactive during this experiment. The authors should perform an additional control experiment showing that nCas9 was active and introduced indels but at the same time did not turn on GFP fluorescence.

We performed an experiment in HEK293.EGFP cells in which we simultaneously targeted the BEAR-mScarlet plasmid with a pegRNA and the genome-integrated GFP sequence with sgRNA(s). This experimental design ensured that the indel-inducing activity of the different transfected SpCas9 variants could be tested simultaneously with their effect on the PEAR plasmid. The results confirmed that the PEAR plasmid reports prime editing activity exclusively and indels introduced by SpCas9 variants such as nCas9 do not turn on mScarlet fluorescence. We also examined the effect of paired nickase and WT-SpCas9 to confirm these conclusions. These results are discussed in the revised manuscript on page 8, lines 164-181 and presented in the revised Figure 1 —figure supplement 1E.

3. The advantage of using prime editing instead of more efficient base editing is that this approach allows the introduction of all types of substitutions and/or precise indels. The authors demonstrated that their reporter system works well when PE is designed to substitute several nucleotides. Is it possible to use this system for designing pegRNAs to perform small or large indels?

We designed twelve additional PEAR-GFP plasmids in which the functional splice site is disrupted such that they can be corrected by insertions (2 constructs) with different lengths and positions, by deletions (7 constructs) with different lengths and positions, or by both insertion and substitution (3 constructs). These constructs showed no fluorescence when transfected into HEK293T cells without PE2. All constructs could be corrected by prime editing with an efficiency of about 20-40%. These experiments showed that it is possible to measure insertions and deletions with the PEAR method. The results are discussed in the revised manuscript on page 8, lines 182-187 and presented in the revised Figure 1C.

Reviewer #2:

I recommend the authors address the following points prior to publication:

(1) As it relates Figure 3b, the standard for analyzing these types of editing events would be an NGS/HTS analysis of the targeted loci to see the individual allelic outcomes from the editing. As in currently stands, the authors only perform Sanger sequencing of the bulk populations. On a related note, does PEAR-based enrichment also increase the frequency of indel formation relative to transfection or no-enrichment? The current Sanger sequencing-based analysis does not provide this sort of insight.

In agreement with the Reviewer, in the revised paper we used NGS to detect prime editing efficiencies and also indels from the same samples. As expected, enriching for prime edited samples also increased the frequency of indel formation to a varying extent but without compromising the specificity of prime editing that we show as the ratio of clean editing / indel for all on-target editing. The corresponding results are presented on Figure 3C-G and Figure 4C-E.

(2) In Figure 3b, the authors do not perform any off-target analysis of prime editor activity. At minimum, the authors should PCR amplify the most-likely off-target loci and confirm that PEAR-based enrichment does not increase likelihood of off-target events. In addition, the authors should ensure that the pegRNA and nicking sgRNA targeting the PEAR plasmids do not induce off-target edits.

Concerning the first part of the recommendation, we analysed prime editing and indel formation on the most likely off-target loci (15 in total) of four genomic targets (HEK 3, HEK4, EMX1 and FANCF). We found no significant off-target prime editing or indel formation (compared to the untransfected control) for 14 out of 15 off-target sites. Moreover, on these sites PEAR enrichment did not increase off-target editing to a detectable level by NGS, while it increased on-target editing by 2.18-4.00 fold.

In case of HEK4 off-target site 3, we observed off-target prime editing and also indel formation. However, PEAR enrichment resulted in a 1.75 specificity ratio versus the 1.26 for the samples without enrichment, meaning that it did not compromise the specificity rather increased it to a small extent. The results are discussed in the revised manuscript on page 15, lines 335-342 and presented in the revised Figure 3F-G.

Regarding the second part of the recommendation, the main problem in evaluating the genome-wide off-targets of the PEAR-pegRNA is that it has no on-target sequence in the genome. Thus, it is not ensured that the lack of off-target editing is due to its high specificity rather than its low efficiency. We solved this problem by choosing GFP target sites which do not have a target in the human genome but could be tested for off-targets in HEK293.EGFP cells using GUIDE-seq. Both EGFP target site 2 (tested by Kulcsár et al., (2020) Figure 5f) and EGFP target site 7 (tested in this work) proved to have no detectable off-target sites. We constructed PEAR-GFP-2in1 plasmids in which the PEAR target sequence was replaced by these targets. We also introduced silent mutations in the GFP coding sequence on the plasmid to prevent the pegRNA from targeting it. Since we did not use a second nicking sgRNA on the plasmid in enrichment experiments, its off-target cleavage has never been a concern. Of the new 2in1 plasmids, the one with the highest editing activity was selected and used for enrichment of successful prime editing at 8 different genomic target sites. The results are discussed in the revised manuscript on pages 14-15, lines 350-378 and presented in the revised Figure 4.

(3) In Figure 3b, the authors perform comparison against reporters of transfection. However, much of the field are using reporters of expression for enriching gene editing events. The authors should make similar comparisons to demonstrate the utility of their method.

We are not convinced that placing the fluorescent reporters to the Cas9 expression plasmid instead of the sgRNA expression plasmid and by joining the Cas9 protein to the fluorescent protein through a 2A tag would change significantly the outcome of the transfection control experiments. Especially, since these types of experiments are affected more by other factors, such as the spectral propensity of the used fluorescent protein and the optical set up of the used FACS machine.

(4) The authors only perform analysis of PEAR in bulk sorted cell populations. Although such enrichment is useful the authors do not provide any analysis of clonally isolated cell populations to determine the utility of PEAR in such applications.

This is another comment, the implementation of which we do not consider necessary to assess the usefulness of PEAR enrichments. Although we agree with the Reviewer that analysis of clonally isolated cell populations would provide additional information, we feel that the information gain is not so substantial that we could afford to do it in the given time frame. We hope that the considerable body of experiments we have done convinces the Reviewer that the manuscript is sufficiently improved by the revision.

(5) The authors only perform analysis on 3 genomic loci which does not provide a good indication of the broad utility of PEAR-based enrichment strategies. It is recommended that the authors demonstrate the utility of PEAR-based enrichment methods in the context of additional loci including those that are recalcitrant to typical PE strategies. In addition, the authors should demonstrate the utility of PEAR in the context of other types of PE-driven edits (i.e. small deletions, insertions) in addition to single base pair changes.

In the revised manuscript, we did PEAR enrichments with 14 different prime editing modifications on 8 additional (11 in total) genomic targets in HEK293T cells. Two of these target sites (HEK site 4 and EMX site 1) are known to be recalcitrant to typical PE strategies. These modifications include substitution (7 targets), deletion (3 targets), insertion (3 targets) and insertion-deletion (1 target) mutations. These 14 prime editing modifications experiments resulted in a 2.12-4.63 fold enrichment compared to the control without enrichment. The results are discussed in the revised manuscript on pages 13-14, lines 308-328 and page 15 lines 373-378 and presented in the revised Figure 3C-E and Figure 4C-E.

(6) Is the fluorescent signal associated with the PEAR plasmids transient? Are there a certain number of passages required to lose the fluorescent signal? Along similar lines, the authors should at least discuss what strategies can be taken to ensure there is not genomic integration of the PEAR reporter plasmids.

In the timeframe of a PEAR experiment (1-3 days after transfection) the signal is expected to be mainly transient. The integration of a circular plasmid without homology arms into the genome is generally very low even at sites of Cas9 induced double stranded DNA breaks (DSB). Without DSB this is expected to be even lower. A previous study found that two weeks is sufficient for N2a cells to lose the fluorescent signal of a plasmid expressing GFP, the time it takes for plasmids and their expressing proteins to degrade completely. (Tálas et al., Sup. Figure S1).

To also address the Reviewer’s concern experimentally, GFP-positive cells from PEAR-enriched populations of 4 different genomic targets were collected by cell sorting. The sorted, 100% GFP-positive cell populations were passaged for 3 and 4 weeks (to ensure that no transient signal remained), and GFP positive cells were measured via flow cytometry. Compared to the control (untransfected cells, 0.02% GFP positive), 0.02-0.05% of cells were found in the GFP gate. These results support that the integration of active PEAR plasmids do not occur frequently, and the signal we see in the first couple of days is fully transient. However, these experiments cannot completely rule out the general, random integration of these plasmids that occurs naturally in experiments with plasmids. Therefore, we have developed PCR primers that can be used to detect plasmid integration. The primers and the PCR protocol is presented in Table 1. We are discussing this issue on page 14 lines 343-349. The results are presented in the revised Figure 3 —figure supplement 1.

(7) The authors only perform PEAR based enrichment with HEK293 cells, which are very amenable to gene editing. To demonstrate the broad utility of this tool the authors should perform additional experiments in other cells lines, including those such a primary cells or pluripotent stem cells which are resistant to genetic modification.

In response to this recommendation, we performed PEAR enrichments on K562 and U2OS cell lines and on a human embryonic stem cell line HUES9 on four, three and one genomic targets, respectively. We detected prime editing efficiencies and also indels from the same samples using NGS. The experiments resulted in 1.66-4.16 fold enrichment in K562, in 1.49-2.71 fold enrichment in U2OS and in 7.86 fold enrichment in the HUES9 cell lines compared to the controls without enrichment. The results are presented in the revised Figure 3C-E.

(8) Throughout the manuscript, information about statistical analysis, number of biological replicates, and other information related to scientific rigor are missing.

Thank you for your comment, we have added all the information on statistics and replications in parallel with the more relevant characterization of our approach.

We would like to thank to the Reviewer for their helpful comments and suggestions that helped us to improve the quality of our manuscript.

Reviewer #3:

Prime editing is an exciting new tool in the CRISPR genome editing toolbox which can introduce targeted sequence insertions, deletions and base substitutions. New strategies to enrich for prime edited cells would abrogate the need for time-consuming single-cell sorting and clonal expansion. Simon and colleagues sought to develop a method for easily recovering prime edited cells. Such a method would be useful, as prime editing thus far has only reached moderate levels of efficiency. To achieve this, the authors developed a fluorescence-on, plasmid-based reporter that, when successfully prime edited, repairs a damaged splice site and leads to expression of a fluorescent transgene.

The authors nicely demonstrate that sorting out fluorescent, reporter-positive cells enriches for successful prime editing in the cellular genome. Prime editing of the surrogate plasmid reporter robustly enriched for successful prime editing in a genomically-integrated transgene. Interestingly, this method was also successful when one pegRNA drove prime editing of the plasmid reporter and one pegRNA directed a single base change in endogenous genes (FANCF, RNF2, HEK3).

One key feature of prime editing is that it can be used to make complex genomic alterations such as templated sequence insertions. This has been used to epitope tag endogenous genes (FLAG/His6 tags) and make prime editing distinct from base editors, which aim to alter single base pairs. A weakness of the work presented by Simon and colleagues is that they limit testing of the PEAR reporter to enrichment of cells following single base pair modifications in endogenous genes. In this context, the PEAR reporter was very good at enriching for prime edited cells but it is an open question as to how well PEAR would allow for the enrichment of more complex prime edits (such as multiple base changes or sequence insertions/deletions). Additionally, it is unclear how well the PEAR reporter would enrich for prime-edited cells edited at low efficiency (<10%), where enrichment would be most valuable.

In the revised version, we did PEAR enrichments with 14 different prime editing modifications on 8 additional (11 total) genomic targets in HEK293T cells. Two of these target sites (HEK site 4 and EMX site 1) are known to be recalcitrant to typical PE strategies. These modifications include substitution (7 targets), deletion (3 targets), insertion (3 targets) and insertion-deletion (1 target) mutations. With these 14 prime editing modifications experiments resulted in 2.12-4.63 fold enrichment compared to the control without enrichment. The results are discussed in the revised manuscript on pages 13-14, lines 308-328 and page 15 lines 373-378 and presented in the revised Figure 3C-E and Figure 4C-E.

Lastly, the PEAR reporter functioned well in the cell line tested (HEK293Ts) but it is unknown how this strategy would work in other immortalized cell lines or primary cells. The PEAR reporter would be a boon if it could successfully enrich for prime edited cell types that are not amenable to single cell sorting and clonal expansion (such as primary cells or cell lines that are difficult to grow up from a single cell, such as Calu-3s).

Unfortunately, our lab is not prepared to demonstrate the method on primary cells or Calu3. However, we performed PEAR enrichments on K562 and U2OS cell lines and on a human embryonic stem cell line, HUES9 on four, three and one genomic targets, respectively. We detected prime editing efficiencies and also indels from the same samples using NGS. The experiments resulted in 1.66-4.16 fold enrichment in K562, in 1.49-2.71 fold enrichment in U2OS and in 7.86 fold enrichment in the HUES9 cell lines compared to the controls without enrichment. The results are presented in the revised Figure 3C-E.

Together, the authors achieved their aim of developing a fluorescence-on reporter that allows for the enrichment of prime edited cells, both when the prime edit is made in a genomically-integrated transgene or at an endogenous genomic site. This tool will be useful for the genome editing community and/or researchers who wish to introduce minimal base pair changes in HEK293T cells.

In the discussion, the authors state that "The tolerance of the 5' splice site for substitutions makes the sequences of the target region of the PEAR plasmid easily adjustable." The manuscript would be strengthened if the authors demonstrate that this is true.

We designed twelve additional PEAR-GFP plasmids in which the functional splice site is disrupted such that they can be corrected by insertions (2 constructs) with different lengths and positions, by deletions (7 constructs) with different lengths and positions, or by both insertion and substitution (3 constructs). These constructs showed no fluorescence when transfected into HEK293T cells without PE2. All constructs could be corrected by prime editing with an efficiency of about 20-40%. These experiments showed that it is possible to measure insertions and deletions with the PEAR method. The results are discussed in the revised manuscript on page 8, lines 182-187 and presented in the revised Figure 1C.

In addition to these experiments where the splice site itself was not modified per se, we have constructed 8 additional plasmids where the splice site, the 5’ flanking sequence or the 3’ flanking sequence was modified around the splice site. These inactive plasmids have shown very low to no fluorescence when transfected to HEK293T cells. Transfecting the corresponding plasmids with active splice sites (generated by molecular cloning) resulted in high percentage of GFP positive cells. When the inactive plasmids were prime edited by PE2 it resulted in 6-71% GFP positive cells indicating that prime editing can correct all constructs with varying efficiencies. The correction of the PEAR plasmid was highly dependent on the strength of the splice site itself, and the number of modified bases. The results are discussed in the revised manuscript on pages 8-9, lines 188-193 and presented in the revised Figure 1D.

We would like to thank to the Reviewer for their helpful comments and suggestions that helped us to improve the quality of our manuscript.

Reviewer #4:

[…]

The following comments are intended to improve the manuscript.

"more precise CRISPR tools; base" … this should not be a semicolon. Consider a pair of dashes, and adjusting "developed, that can" to "developed, and these enzymes can"

"BEs'." no apostrophe needed; "of BEs" conveys the possessive.

Thank you for catching these. In the revised manuscript it reads as:

page 3, lines 29-31: “Recently, more precise CRISPR tools – base ^89, and prime ¹⁰ editors – have been developed, and these enzymes can introduce modifications into the DNA”

page 3, line 35: “lags behind that of BEs”

Figure 1e: the black text can be hard to see when it is in front of the black data points.

In the revised manuscript we removed the flow cytometry plot from Figure 1 and placed it to Figure 1 —figure supplement 1D as we wanted to show example flow cytometry plots from each major experiment. In the revised figure all numbers are visible.

"former observations that indels cannot, only substitution mutations" … This seems incorrect.

We have removed this line and the corresponding figure from the revised manuscript and replaced it with Figure 1 —figure supplement 1E. Which we discuss on page 8, lines 164-181. The conclusion is now as follows:

“…PEAR specifically reports on prime editing activity and is insensitive to the potential presence of indel background…”

We would like to thank to the Reviewer for their helpful comments and suggestions that helped us to improve the quality of our manuscript.

References

Kulcsár, P.I., Tálas, A., Tóth, E. et al. Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs. Nat Commun 11, 1223 (2020). https://doi.org/10.1038/s41467-020-15021-5

Tálas, András et al. “A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.” DNA Research vol. 24,6 (2017): 609-621. doi:10.1093/dnares/dsx029

Author details

1. Dorottya Anna Simon

   1. Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
   2. ProteoScientia, Budapest, Hungary
   3. School of Ph.D. Studies, Semmelweis University, Budapest, Hungary

   Contribution

   Data curation, Formal analysis, performed experiments and interpreted the results

   Contributed equally with

   András Tálas

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0252-0072

2. András Tálas

   Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary

   Contribution

   Conceptualization, Data curation, Formal analysis, Writing – original draft, conceived and designed the experiments, contributed to molecular cloning, performed experiments on mammalian cells, interpreted the results and wrote the manuscript with input from all authors

   Contributed equally with

   Dorottya Anna Simon

   For correspondence

   talas.andras@ttk.hu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0851-8333

3. Péter István Kulcsár

   1. Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
   2. Biospiral-2006, Szeged, Hungary

   Contribution

   Data curation, Formal analysis, contributed to molecular cloning, experiments with U2OS cells and executed GUIDE-seq

   Competing interests

   No competing interests declared

4. Zsuzsanna Biczók

   1. Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
   2. School of Ph.D. Studies, Semmelweis University, Budapest, Hungary

   Contribution

   Data curation, performed experiments with stem cells

   Competing interests

   No competing interests declared

5. Sarah Laura Krausz

   1. Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
   2. School of Ph.D. Studies, Semmelweis University, Budapest, Hungary

   Contribution

   Software, analysed NGS data

   Competing interests

   No competing interests declared

6. György Várady

   Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary

   Contribution

   Data curation, Resources, performed cell sorting

   Competing interests

   No competing interests declared

7. Ervin Welker

   1. Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
   2. Institute of Biochemistry, Biological Research Centre, Szeged, Hungary

   Contribution

   Formal analysis, Supervision, Writing – original draft, conceived and designed the experiments, interpreted the results, supervised the research and wrote the manuscript with input from all authors

   For correspondence

   welker.ervin@ttk.hu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9874-8794

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