MainBase editors1,2 are a transformative genome-editing technology that can precisely install single-base alterations into cellular genomic DNA (gDNA) without requirements for DNA double-strand breaks (DSBs) or exogenous DNA donor templates or reliance on cellular homology-directed repair pathways. For example, adenine base editors (ABEs) and cytosine base editors (CBEs) are capable of reverting G•C to A•T and T•A to C•G mutations, respectively, which represent the most common pathogenic single-nucleotide variants reported in the ClinVar database (~47% and 14%)3, highlighting the enormous potential of this class of genome editors for therapeutic application.However, like other genome editors, base editors can bind and modify off-target genomic loci with sequence homology to the target protospacer4,5,6. As base editors rapidly advance toward clinical applications, defining guide RNA (gRNA)-dependent off-target activity of base editors in a sensitive and unbiased way is important for assessing the safety of these new therapeutic approaches7. A method that is unbiased is one that does not preselect candidate off-target sequences for experimental analysis.Current methods for profiling the genome-wide activity of base editors8,9,10 have certain limitations. Digenome-seq8 and EndoV-seq9 are based on whole-genome sequencing (WGS) of base-editor-treated gDNA in vitro, followed by processing with end repair enzymes to generate DSBs detectable by high-throughput sequencing. As these methods do not enrich for base-editor-modified gDNA, they require hundreds of millions of sequencing reads, reducing sensitivity and scalability and increasing costs. The apparent validation rate of these methods for identifying bona fide cellular off-target base-editing mutations is relatively low8,9 and the high sequencing depth requirements limit their application to many targets. Another technique, ONE-seq, relies on biased computational preselection of candidate off-target sites and, therefore, cannot detect genome-wide off-target activity beyond those sites selected a priori.Sensitive and unbiased approaches offer the unique advantage of detecting unintended on-target genome editor off-target activity caused by ‘unknown unknowns’ such as contaminant gRNAs.In earlier studies, we and others applied indirect methods to identify base editor off-target activity11,12,13,14,15, relying on the simplifying but not necessarily true assumption that base editor off-target effects are a subset of nuclease off-target effects. For example, to experimentally nominate candidate off-target sites for an ABE therapeutic strategy to treat sickle cell disease, we performed CIRCLE-seq16,17 (an earlier method we developed for selective sequencing of Cas9-cleaved gDNA) using Cas9 nuclease to nominate candidate off-target sites. We then measured editing activity at CIRCLE-seq Cas9-nominated off-target sites in a base-editing context in CD34+ hematopoietic stem and progenitor cells (HSPCs) treated with ABE8e-NRCH:HBBS-gRNA using multiplex targeted sequencing. We identified 54 Cas9-nuclease-nominated off-target sites with unintended off-target base-editing activity as high as 82%11.Although gRNA-dependent base-editing off-target sites often overlap with Cas9 nuclease off-target sites8,9,18, productive base editing requires additional criteria, such as the presence of the target base within the editing window, base edited nucleotide sequence context and R-loop accessibility by deaminases, which may not be satisfied for all Cas-nuclease-dependent off-target sites19. Thus, limitations of our initial nuclease-centric approach are that base editors may have a different spectrum of off-target activity than cognate Cas9 nucleases, highlighting an unmet need for a simultaneously sensitive and unbiased method for specifically defining base editor genome-wide off-target activity. Unbiased methods to characterize gene therapy products that use base editing to gain regulatory approval for clinical trials should be performed with the relevant base editor and not a similar nuclease.Previously, we developed CHANGE-seq20, a sensitive, unbiased and high-throughput method to identify Cas9 nuclease off-target activity in vitro. CHANGE-seq leverages a Tn5 tagmentation-based workflow for efficient generation of circularized gDNA libraries, coupled with stringent selection of circular DNA molecules using a cocktail of exonucleases, thereby generating a population of circular DNA molecules with minimal free DNA ends. Covalently closed circular DNA molecules are treated with CRISPR–Cas9 ribonucleoprotein (RNP) complexes and nuclease-cleaved DNA fragments are then ligated to adaptors and sequenced, enabling the selective sequencing of Cas9-cleaved DNA molecules.Here, we present CHANGE-seq-BE, a method for both sensitive and unbiased genome-wide discovery of gRNA-dependent base editor on-target and off-target sites, adapted from CHANGE-seq. After extensive molecular biology optimization, we overcame substantial technical challenges in enriching for editor-modified DNA reported by us and others10,20. CHANGE-seq-BE now enables the selective and efficient sequencing of base-editor-modified gDNA for both ABEs and CBEs.We applied CHANGE-seq-BE to define the gRNA-dependent genome-wide off-target activity of ABE8e and CBE eA3A-BE3 at therapeutically relevant genes in primary human cells. We found that CHANGE-seq-BE sensitively identifies not only off-target activity but also unintended on-target activity of contaminating synthetic gRNAs, highlighting one of the advantages of using unbiased sensitive methods for genome-wide assessment of genome-editing activity. In direct comparisons of ABEs and Cas9 nuclease at the same targets, we found substantially higher off-target editing activity with ABE8e base editors. Lastly, we used CHANGE-seq-BE to support genotoxicity studies in an emergency investigational new drug (IND) application for an adenine-base-editing treatment for a person with CD40L-deficient X-linked hyper IgM (X-HIGM) syndrome.ResultsDevelopment of a sensitive and unbiased biochemical method to measure base editor genome-wide activityMotivated by a critical need for better methods to directly characterize the gRNA-dependent genome-wide off-target activity of base editors, we sought to develop a method that would be simultaneously sensitive, unbiased, sequencing efficient and straightforward to practice. We reasoned that CHANGE-seq could be adapted for base editors by treating purified, circularized gDNA with base editor RNP complexes and selectively sequencing base-editor-modified gDNA (Fig. 1a). First, linear gDNA could be circularized using our previously described CHANGE-seq Tn5 tagmentation-mediated approach20. Second, for ABEs, enzymatically purified gDNA circles could be treated with ABE RNP complexes to nick the target DNA strand and deaminate adenine bases to inosine (within the base-editing window) on the nontarget strand at on-target and off-target sites. Third, nicked and inosine-containing gDNA circles could be further processed with endonuclease V (ref. 21), which cleaves DNA adjacent to inosines to produce linear DNA with 5′ staggered ends. Similarly, for CBEs, circularized gDNA could be treated with CBE RNP complexes to nick the target DNA strand and deaminate cytosine bases to uracil on the complementary DNA strand. Uracil-containing gDNA circles could be further processed with USER enzyme (uracil-specific excision reagent), a mixture of uracil DNA glycosylase and the DNA glycosylase–lyase endonuclease VIII, which cleaves and removes uracil in the nontarget DNA strand to generate linearized DNA. The base-editor-modified linear DNA processed by endonuclease V or USER enzyme could be end-repaired and ligated to sequencing adaptors for selective high-throughput sequencing of base-editor-modified gDNA molecules.Fig. 1: Development of CHANGE-seq-BE, a sensitive and unbiased biochemical method to detect base editor genome-wide activity.a, Schematic of CHANGE-seq-BE workflow. Purified gDNA is tagmented with custom Tn5 transposome containing circularization adaptor DNA. Tagmented gDNA is circularized by intramolecular ligation. Residual linear DNA is enzymatically degraded with an exonuclease cocktail, leaving highly pure gDNA circles. Circularized gDNA is treated in vitro with ABE or CBE RNP complexes to nick the target DNA strand and deaminate adenine bases to inosine or cytosine bases to uracil (within the base-editing window) on the nontarget strand at on-target and off-target sites (red). For ABEs, nicked inosine-containing gDNA circles are then processed with endonuclease V, which cleaves DNA adjacent to inosines. For CBEs, uracil-containing DNA circles are treated with USER, which excises the uracil base to produce linear DNA with 5′ staggered ends. The 5′ overhangs are filled in with Klenow exo-, end-repaired, ligated to sequencing adaptors and amplified for selective high-throughput sequencing. b, Visualization of detected off-target sites identified by CHANGE-seq-BE aligned against the intended target site for ABE:gRNA (ABE8e-NRCH:HBBS-gRNA) complexes targeted against HBB. The intended target sequence is shown in the top line and off-target sites are ordered from top to bottom by CHANGE-seq-BE read count. Mismatches to the intended target sequence are indicated by colored nucleotides; matches are shown as dots and read counts and A-to-I (G) deamination frequencies (%) are shown at the end of each line (only the top off-target sites are listed). Representative alignment of CHANGE-seq-BE reads of the ABE8e-NRCH:HBBS-gRNA target, as visualized by the Integrative Genomics Viewer (IGV) for an off-target site on chr2. The target adenine base is highlighted in red, the protospacer-adjacent motif (NRCH) is shown in bold and the single-stranded DNA breaks are marked with colored arrows by nCas9 (purple) and endonuclease V (red). c, Visualization plot of off-target sites aligned against the intended target site for selected eA3A-BE3:RNF2 complexes. The intended target sequence is shown in the top line and off-target sites are ordered by descending CHANGE-seq-BE read count. Mismatches to the intended target sequence are indicated by colored nucleotides and the arrow indicates the on-target site. Alignment of CHANGE-seq-BE reads of the eA3A-BE3-NGG:RNF2 gRNA off-target site on chr17, as visualized by the IGV. The target cytosine base in the editing window is highlighted in red, the protospacer-adjacent motif (NGG) is depicted in bold and single-stranded breaks made by USER (red) and nCas9 (purple) are indicated with respective colored arrows.Full size imageHowever, a major challenge we initially encountered was that the addition of the required end-repair step substantially decreased the enrichment of editor-modified DNA20. To gauge the performance of our initial CHANGE-seq for base editor approach, we quantified the number of validated off-target sites detected for an ABE8e RNP targeting the HBB gene (ABE8e-NRCH:HBBS-gRNA) that we previously characterized extensively and validated 54 off-target sites11. At first, we found that addition of an end-repair step to fill in 5′ overhangs and detect base-editor-modified gDNA resulted in high background and detection of only a small fraction (20%) of previously validated off-target sites (Extended Data Fig. 1a). We hypothesized that residual linear DNA molecules could become competent for adaptor ligation after end repair. After systematic optimization of the entire workflow, where we varied circularized gDNA exonuclease treatments, base-editing reaction conditions and end-repair protocols, we identified an optimized protocol (Supplementary Note 1 and Supplementary Protocol) that consistently detected all 54 previously validated ABE8e-NRCH:HBBS-gRNA off-target sites, which we called CHANGE-seq-BE (Extended Data Fig. 1a).For ABEs, CHANGE-seq-BE reads capture a distinct molecular signature of base editing in vitro, evidence of A-to-I deamination as inosine bases are converted to guanine after PCR amplification, increasing confidence in sites nominated by our method. We observed a range of deamination frequencies of 25–48% in top ABE8e-NRCH:HBBS-gRNA off-target sites nominated by CHANGE-seq-BE (Fig. 1b).CHANGE-seq-BE is also readily adaptable to CBEs, as long as high-quality, purified CBE proteins with efficient biochemical activity are available. To evaluate the activity of CBEs, we first performed in vitro cleavage profiling of a PCR amplicon using AnBE4max, A3A-BE3, eA3A-BE3, Td-CGBE and Td-CBEmax22,23,24 (Extended Data Fig. 1b) RNP complexes, followed by USER treatment to create DSBs. On the basis of protein activity, quality and purity, we selected eA3A-BE3 that showed 95% cleavage activity to further test with CHANGE-seq-BE. In contrast to ABEs, the USER enzyme recognizes and excises deaminated uracil bases from the noncomplementary strand and, thus, C-to-T base conversion is not detectable in CHANGE-seq-BE reads, although the breakpoint positions remain clear (Fig. 1c).CHANGE-seq-BE identifies new bona fide off-target sites for ABE8e-NRCH:HBB S targetTo initially evaluate technical reproducibility, we performed independent CHANGE-seq-BE library preparations and found that read counts from technical replicates were strongly correlated (Pearson’s correlation coefficient, r = 0.9814) (Fig. 2a), with sites of off-target activity distributed broadly throughout the genome (Fig. 2b and Supplementary Table 1). For this gRNA target, the on-target site did not have the highest CHANGE-seq-BE read counts, consistent with the lower specificity profile we observed.Fig. 2: CHANGE-seq-BE sensitively reveals new bona fide off-target sites for ABE8e-NRCH:HBBS-gRNA therapeutic relevant target site.a, Scatter plot showing read count correlation for two CHANGE-seq-BE library preparations of ABE8e-NRCH targeting HBBS. b, Manhattan plot showing the genome-wide distribution of CHANGE-seq-BE-detected sites for ABE8e-NRCH:HBBS-gRNA. The pink arrow indicates the on-target site. c, Venn diagram showing overlap between candidate off-target sites nominated by CHANGE-seq-BE for ABE8e-NRCH:HBBS-gRNA and nominated off-target sites for which off-target editing was observed by multiplex targeted sequencing in CD34+ cells (from a person with sickle cell disease) nucleofected with ABE8e-NRCH mRNA. d, Dot plot showing the percentage of sequencing reads containing A-to-G base editing within the protospacer positions 4–10 at off-target sites in gDNA samples derived from CD34+ HSPCs treated with ABE8e-NRCH mRNA (red, n = 2), or untreated controls (gray, n = 2). χ2 tests were performed between mRNA-treated and control samples. The FDR was calculated using the Benjamini–Hochberg method. Reported off-target sites were considered statistically significant with FDR ≤ 0.05 and difference ≥0.5% between treated and untreated controls. e, Dot plot showing the enrichment ratio for sites detected by CHANGE-seq-BE using ABE8e or for CIRCLE-seq using cognate Cas9 nuclease. Statistical analysis was conducted using an unpaired t-test (two-tailed) with P = 0.0098.Full size imageTo determine whether CHANGE-seq-BE identifies base editor-specific off-target activity not detected by nuclease-specific methods, we compared the sites identified by CHANGE-seq-BE and CIRCLE-seq using ABE8e-NRCH base editor and Cas9-NRCH nuclease, respectively. We noted that CHANGE-seq-BE and CIRCLE-seq read counts were only weakly correlated (r = 0.34) (Extended Data Fig. 2a) and only 40% of the off-target sites detected by both methods overlapped (Extended Data Fig. 2b), suggesting that ABE8e genome-wide off-target activity quantitatively differs from Cas9 nuclease off-target activity. One explanation for some of these differences is the number and location of editable adenine bases within on-target and off-target sites, as off-target sites with mismatches of the editable adenine bases could eliminate base-editing activity without affecting nuclease activity.To confirm that new off-target sites detected exclusively by CHANGE-seq-BE are bona fide off-target sites in cells, we performed multiplex targeted sequencing on 131 sites detected exclusively by CHANGE-seq-BE (Supplementary Table 2) using the same gDNA of CD34+ HSPCs treated with ABE8e-NRCH mRNA evaluated in our previous study11. We found that CHANGE-seq-BE identified all 54 known cellular off-target sites, as well as an additional 29 previously unknown bona fide off-target sites, 53% more than our initial screen with CIRCLE-seq (Fig. 2c), with ABE activity ranging from 0.55% to 13.9% (Fig. 2d). For the HBBS-gRNA bona fide off-target sites identified by CIRCLE-seq using Cas9 nuclease and CHANGE-seq-BE using ABE8e, we noted that the CHANGE-seq-BE enrichment ratio was significantly higher than that of CIRCLE-seq, by an average of 2.1-fold (Fig. 2e). HBBS-gRNA bona fide off-target activity occurred primarily in intergenic and intronic regions and six off-target sites were in exons (Extended Data Fig. 2c). Taken together, our results demonstrate the importance of using sensitive and unbiased base-editor-specific off-target discovery methods to comprehensively identify ABE genome-wide off-target activity.CHANGE-seq-BE sensitively identifies bona fide off-target sites in human primary cellsTo systematically assess the genome-wide off-target activity of ABEs and CBEs in vitro, we performed CHANGE-seq-BE on five therapeutically relevant target sites in human primary T cells (B2M, CBLB, CD7, CIITA and PDCD1)25 and in human primary hepatocytes (PCSK9)26 with ABE8e (Fig. 3a and Extended Data Fig. 3a). Overall, CHANGE-seq-BE nominated a range of 236 to 1,604 on-target and off-target sites (total of 4,199) for B2M, CBLB, CD7, CIITA, PDCD1 and PCSK9 across the genome in two experimental replicates (Fig. 3b, Extended Data Fig. 3b and Supplementary Table 3).Fig. 3: CHANGE-seq-BE detects bona fide base editor off-target sites for ABE8e in primary human T cells.a, Schematic of CHANGE-seq-BE off-target nomination at five targets in primary human T cells (B2M, CBLB, CD7, CIITA and PDCD1). b, Manhattan plot showing the genome-wide distribution of CHANGE-seq-BE-detected sites. The arrow (pink) indicates the on-target site. c, Venn diagrams depicting the overlap of off-target sites nominated by CHANGE-seq-BE and Digenome-seq and validated sites by hybrid capture sequencing. d, Bona fide cellular off-target sites detected by hybrid capture sequencing for the sites nominated by CHANGE-seq-BE and Digenome-seq, including CHANGE-seq-BE read counts (columns). Dot plot showing the percentage of sequencing reads containing A-to-G mutations within the protospacer positions 1–10 at off-target sites in gDNA samples from primary human T cells treated with ABE8e-NGG mRNA (red, n = 3) or untreated controls (gray, n = 3). χ2 tests were performed between mRNA-treated and control samples. The FDR was calculated using the Benjamini–Hochberg method. Reported off-target sites were considered statistically significant with FDR ≤ 0.05 and difference ≥0.5% between treated and untreated controls. e, Stacked bar plot representing the number of validated off-target sites exclusively identified by CHANGE-seq-BE (yellow), Digenome-seq (purple) or both (blue). f, Genomic features of validated off-target sites for six ABE targets (B2M, CBLB, CD7, CIITA, PDCD1 and PCSK9). TTS, transcription termination site; UTR, untranslated region. The proportion of transcribed regions in the validated sites was statistically significant compared to CHANGE-seq-BE-nominated sites using Fischer’s exact test (two-sided) with P = 0.0264. Panel a created with BioRender.com.Full size imageTo compare the sensitivity of CHANGE-seq-BE to another leading off-target discovery method, we performed Digenome-seq on the same gRNA targets and evaluated them with matching analysis parameters. We identified a total of 796 on-target and off-target sites (ranging from 36 to 162 sites) for B2M, CBLB, CD7, CIITA and PDCD1. Next, we comprehensively evaluated 4,291 sites by hybrid capture sequencing for the sites nominated by either of the methods on gDNA obtained from edited cells compared to unedited controls. At these sites that were successfully captured and amplified, 96 sites showed significant off-target alterations (Supplementary Table 4), with cellular activity ranging from 0.44% to 93.1% (Fig. 3c,d and Extended Data Fig. 3c,d). At 83 (of 96) bona fide T cell off-target sites, 33.3–100% of sites were identified by both methods, 6.9–16.7% of sites were identified by Digenome-seq and 29.3–55.6% of sites were identified exclusively by CHANGE-seq-BE (Fig. 3e). Additional analysis of off-target sites with relaxed parameters found an additional six validated sites (Supplementary Note 2 and Supplementary Table 5).To better understand the functional consequences of off-target activity for ABE targets, we annotated and analyzed the genomic locations of off-target sites by CHANGE-seq-BE and Digenome-seq. The proportion of nominated off-target sites found across various genomic regions (intronic, intergenic, transcribed and promoter) was similar between CHANGE-seq-BE and Digenome-seq (Extended Data Fig. 3e). Both nominated and validated off-target sites occurred predominantly in intergenic and intronic regions, consistent with their abundant representation within the human genome. Compared to the proportion of CHANGE-seq-BE-nominated off-target sites (10.3%), validated cellular off-target sites (17.7%) were significantly enriched in transcribed regions (Fig. 3f).Similarly, we performed CHANGE-seq-BE and Digenome-seq using eA3A-BE3 to target B2M, CBLB, CD7, CIITA and PDCD1 regions at different loci. We identified a total of 850 on-target and off-target sites (ranging from 61 to 198) by CHANGE-seq-BE and 25 sites (ranging from 3 to 11) by Digenome-seq for B2M, CBLB, CD7, CIITA and PDCD1 targets, respectively (Fig. 4a,b and Supplementary Table 6). By hybrid capture sequencing, we evaluated 691 sites nominated by CHANGE-seq-BE and Digenome-seq for all five gRNA targets. Both methods identified on-target editing for all targets with cellular activity ranging from 52.3% to 97.6%. Both methods also detected a high-frequency off-target site (OT01) for CIITA that has a wobble mismatch to the intended target with 17% off-target editing. However, many low-frequency off-target sites with editing rates ranging from 1.7% to 7.5% were not identified by Digenome-seq and were only discovered by CHANGE-seq-BE (Fig. 4c and Supplementary Table 7).Fig. 4: CHANGE-seq-BE detects bona fide off-target sites for eA3A-BE3 in primary human T cells.a, Manhattan plot showing the genome-wide distribution of off-target sites detected by CHANGE-seq-BE for the targets B2M, CBLB, CD7, CIITA and PDCD1. The arrow (pink) indicates the on-target site. b, Venn diagram depicting the overlap of off-target sites detected by CHANGE-seq-BE, Digenome-seq and validated off-target sites. c, Bona fide cellular off-target sites by hybrid capture sequencing for the sites nominated by CHANGE-seq-BE and Digenome-seq, including CHANGE-seq-BE read counts (columns) with sequences. Dot plot showing the percentage of sequencing reads containing C-to-any mutations within the protospacer positions 1–10 at off-target sites in gDNA derived from primary human T cells treated with eA3A-BE3 mRNA (red) or untreated controls (gray) (n = 2). χ2 tests were performed between mRNA-treated and control samples. The FDR was calculated using the Benjamini–Hochberg method. Reported off-target sites were considered statistically significant with FDR ≤ 0.05 and difference ≥0.5% between treated and controls.Full size imageIn sum, our data show that CHANGE-seq-BE is more sensitive than Digenome-seq while requiring 20-fold fewer sequencing reads. CHANGE-seq-BE is a strong predictor of base editor cellular off-target activity in human primary cells and can be used to select candidate target sites for both routine and therapeutic applications.CHANGE-seq-BE identified unexpected synthetic gRNA contaminantsSafety risks specifically associated with human genome-editing products include unintended on-target and off-target activity and their long-term consequences. One less obvious but key concern is that contamination in a critical genome-editing component such as the gRNA could direct genome editing to unintended on-target sites. Chemically modified synthetic gRNAs are now a commonly used product in genome-editing experiments, particularly in primary cells, because of enhanced activity and stability27.In initial pilots of CHANGE-seq-BE experiments using research-grade, chemically synthesized and high-performance liquid chromatography (HPLC)-purified gRNAs, we observed CHANGE-seq-BE read counts across multiple targets in base-editor-treated samples but not in untreated controls, suggesting activity of contaminating synthetic gRNAs (Fig. 5a). We detected strong, unintended activity at PDCD1, CD7 and CIITA target sites in gDNA samples treated with the CBLB gRNA target site; similarly, PDCD1 and CD7 base editing mutation-containing reads were found in gDNA samples treated with CIITA gRNA and B2M and CD7 unintended on-target edited reads were found in gDNA samples treated with PDCD1 gRNA. In hindsight, according to manufacturer discussions, the most likely source of this cross-contamination during manufacturing was the sequential purification of these gRNAs over the same HPLC column. Detectable cross-contaminating activity was mitigated by a more stringent, column clearing-in-place procedure used in a second batch of gRNAs from the same manufacturer.Fig. 5: CHANGE-seq-BE identifies activity of synthetic gRNA contaminants.a, Bar plot showing CHANGE-seq-BE read counts for three synthetic chemically modified gRNAs (CBLB, CIITA and PDCD1) from the manufacturer supplier A (batch 1) and the unexpected detection of gRNA contaminants (red). Data are shown as the mean (n = 2). b, SMARTer gRNA-sequencing workflow. c, Alignment plot for CBLB gRNA target displaying top 20 mapped reads. Mismatched nucleotides in the target sequence are highlighted in colors (blue, green, red and yellow) representing potential contaminants CIITA and PDCD1. The arrow highlighted in the scaffold region (green) shows the scaffold specific primer used for first cDNA synthesis. d, Bar plot showing the percentage of sequencing reads mapping to target gRNAs, for one specific synthetic gRNA supplier A (batch 1). The activity of gRNA contaminants is shown in red. e, Bar plot showing the percentage of sequencing reads containing A-to-G mutations within the protospacer positions 4–10 at off-target sites in gDNA samples from primary human T cells treated with ABE8e in the mRNA format and gRNAs targeting CBLB or CIITA, as well as the detection of contaminant gRNAs (red) during the manufacture process. Data are shown as the mean. Unpaired t-tests (two-sided) were performed using the Holm–Šídák multiple-comparisons method (n = 3); adjusted P = 0.0013 (CBLB on-target), 0.0013 (PDCD1), 0.00002 (CIITA), 0.86) and CHANGE-seq-BE (r > 0.96) (Extended Data Fig. 5a,b). However, across methods, CHANGE-seq and CHANGE-seq-BE read counts were less correlated and varied depending on the gRNA target (r = 0.2–0.73) (Fig. 6a). Our results show that the biochemical activities of ABE8e base editor and Cas9 are both highly reproducible and distinct.Fig. 6: Biological differences in in vitro and cellular off-target activity between nucleases and base editors.a, Scatter plot showing read counts correlation between CHANGE-seq-BE and CHANGE-seq for five ABE targets (B2M, CBLB, CD7, CIITA and PDCD1). b, Sequences of bona fide validated off-target sites for five gRNA targets in hybrid capture sequencing. The heat map represents the percentage of A-to-G editing by ABE8e base editor or indels by Cas9 nuclease (hybrid capture sequencing) or normalized off-target reads relative to on-target reads (GUIDE-seq-2). Cas9-mediated cellular off-target sites are highlighted in red box. χ2 tests were performed between mRNA-treated and control samples. The FDR was calculated using the Benjamini–Hochberg method. Reported off-target sites were considered statistically significant with FDR ≤ 0.05 and difference ≥0.5% between treated (red box) and controls. c, Stacked bar plot representing the total of number of validated off-target sites for ABE8e and Cas9 mRNA edited T cells.Full size imageTo directly assess the cellular off-target activity differences between Cas9 nuclease and ABEs, we edited T cells for all five targets with Cas9 mRNA and performed hybrid capture targeted sequencing on a total of 2,226 off-target sites (including 1,436 off-target sites nominated only by Cas9 CHANGE-seq). High-frequency on-target editing was achieved for both ABE8e (96.5–99.4% A-to-G base editing; Fig. 3d) and Cas9 (70.7–92% indels) for all five targets. A total of 83 bona fide T cell off-target sites with base editing frequencies ranging from 0.44% to 93.1% were identified for ABE8e across all five targets. In contrast, only one bona fide off-target site with an average of 2.4% indels was confirmed for Cas9 targeting PDCD1 (Fig. 6b,c).To independently confirm the editing activity measured by hybrid capture sequencing, we performed GUIDE-seq-2 (ref. 29) with Cas9 delivered as a mRNA for all five targets (Extended Data Fig. 6a). Under matching delivery conditions, we observed GUIDE-seq-2 off-target activity profiles that were consistent with our hybrid capture targeted sequencing results (Fig. 6b), where only OT01 for PDCD1 was clearly above the lower limits of detection of GUIDE-seq-2 (Extended Data Fig. 6b). GUIDE-seq-2 with Cas9 RNP resulted in slightly more but still fairly low off-target activity, ranging from 0.01% to 3.4%.Overall, base editors have different off-target editing profiles when directly compared to cognate nucleases. For example, our comparisons of Cas9 and ABE8e under controlled delivery conditions demonstrate that ABE8e has significantly increased off-target activity compared to Cas9, with 98.8% of validated off-target sites unique to ABE8e. Therefore, it is essential to use direct methods to evaluate the off-target activity of base editors rather than relying on nuclease-based methods only.CHANGE-seq-BE supports emergency IND for personalized genome-editing treatmentWe applied the sensitive off-target detection capability of CHANGE-seq-BE to quantitatively characterize the genome-wide gRNA-dependent off-target profile of an ABE base-editing strategy to treat a person with X-HIGM syndrome by correcting the CD40L mutation (c.658C>T; p.Q220X). Using CHANGE-seq-BE, we identified a total of 81 potential off-target sites across the human genome and experiments were well correlated across two technical replicates (Extended Data Fig. 7a,b). Most of these off-target sites occurred in intronic and intergenic regions and in two exonic regions (Extended Data Fig. 7c). With confirmatory testing by multiplex targeted sequencing using rhAmpSeq, we observed on-target editing of 95.4% with no detectable off-target effects above the limit of threshold compared to controls (Extended Data Fig. 7d). This successfully supported an emergency personalized base editor hematopoietic stem cell and T cell treatment of a person with CD40L HIGM syndrome (NCT06959771), attesting to the IND-enabling role of CHANGE-seq-BE.DiscussionHere, we described CHANGE-seq-BE, which enables a simultaneously sensitive and unbiased approach for comprehensive profiling of the genome-wide activity of base editors. CHANGE-seq-BE is a way of directly identifying gRNA-dependent base editor off-target effects that combines the desirable qualities of high sensitivity with minimal experimental bias within a single protocol and works well with both ABEs and CBEs. In this study, we used CHANGE-seq-BE to support an emergency IND application.Our approach has some limitations. Firstly, CHANGE-seq-BE libraries currently require higher sequencing depth to achieve similar on-target read counts to CHANGE-seq libraries for nucleases. This may be associated with gRNA-independent deamination on both DNA strands or other technical factors, which we plan to explore and optimize further. Secondly, another limitation shared with other biochemical methods is a low apparent validation rate. Future studies, perhaps leveraging artificial intelligence and machine learning, may help to understand and predict the subset of candidates nominated by biochemical methods most likely to be validated in cells. Thirdly, an intrinsic limitation of all biochemical approaches to characterize the genome-wide activity of editors is the requirement for purified proteins. More base-editing proteins are becoming available commercially and, with optimization, miniaturized, high-throughput protein purification approaches may enable characterization of more engineered base editor variants or orthologs. Lastly, CHANGE-seq-BE is not designed to detect gRNA-independent off-target effects, which are more suitably measured in relevant cellular contexts. This may be important to consider for evolved variants that also exhibit higher gRNA-independent deamination activity30.CHANGE-seq-BE has key advantages over existing methods in terms of sensitivity and sequencing efficiency. In contrast to biochemical methods such as Digenome-seq that rely on WGS of base-editor-modified gDNA, CHANGE-seq-BE has a substantially reduced rate of observed background reads, enabling it to be ~20-fold more sequencing efficient while simultaneously achieving higher sensitivity. The relative sequencing efficiency, requiring ~25 million reads per sample, makes CHANGE-seq-BE accessible to most labs with access to short-read high-throughput sequencers, while enabling the evaluation of more targets in a single sequencing run and the rapid identification of highly active and specific targets. We previously automated CHANGE-seq20 and expect that CHANGE-seq-BE should be similarly automatable.Another advantage is that CHANGE-seq-BE enables direct profiling of base editor activity, rather than relying on the false assumption that nuclease and base editor activities are the same. It is critical to use proteins with the same amino acid sequence to that used during the clinical editing process to assess off-target risks in therapeutics.Our findings clearly show that nuclease and base-editing off-target activity differ significantly in both biochemical and cellular contexts. We found that ABE8e induced more detected off-target sites and considerably higher frequencies of off-target editing activity compared to Cas9 under controlled cellular delivery conditions. This increased off-target activity is likely associated with the 540–1,000-fold increase in DNA deaminase activity in comparison to earlier-generation ABEs30,31 and updates the dogma in the field that base editors are generally more specific than nucleases.CHANGE-seq-BE is relevant for IND-enabling characterization of clinical genome-editing approaches. One example in this study is that we successfully used CHANGE-seq-BE to nominate candidate off-target sites, clear an IND application and treat a person with X-HIGM syndrome with a base editor designed to correct their disease-causing mutation.One interesting observation of our study was that gRNAs with an intended target sequence could be contaminated with others during routine chromatography steps during synthesis and these low-frequency contaminants resulted in measurable activity in biochemical assays such as CHANGE-seq-BE, as well as activity in cells. This unintended ‘on-target’ activity of contaminant gRNAs will be important to consider and measure whenever synthetic gRNAs are used. For research purposes, it may be advisable to avoid sequential HPLC purification of synthesized gRNAs unless stringent procedures to avoid cross-contamination are applied.A general challenge is the functional interpretation of potential base editor off-target sites. For example, it is not technically straightforward to perturb off-target sites individually without affecting other closely related sites and, thus, difficult to derisk them. Thus, although CHANGE-seq-BE represents an important advance to nominating base editor off-target sites, complementary approaches will be required to dissect their function.Once better understood, several strategies can be applied to mitigate deleterious off-target effects and improve the safety and precision of base editors. Previous studies reported that high-fidelity versions of base editors can minimize off-target mutations30,32,33. Alternatively, chimeric DNA–RNA hybrid gRNAs34 offer a promising strategy for mitigating off-target activity without changing the editor protein, although the rules for designing them are not well understood. A recent study by Whittaker et al. demonstrated that hybrid gRNAs can significantly reduce off-target effects including bystander edits while maintaining high on-target editing efficiency35.Overall, with favorable properties of high sensitivity and sequencing efficiency, we anticipate that CHANGE-seq-BE will become increasingly broadly applied36 as an unbiased discovery method to characterize the genome-wide gRNA-dependent off-target activity of base editors for life sciences research and clinical applications.MethodsIsolation of human primary T cellsResearch-consented and deidentified peripheral blood mononuclear cells (PBMCs) were obtained commercially (Key Biologics); CD4+CD8+ T cells were purified using magnetic separation on a CliniMACS Plus instrument. Cells were washed in CliniMACS buffer with 0.5% HSA and resuspended in 190 ml of intravenous immunoglobulin, followed by incubation for 15 min. Subsequently, cells were incubated and labeled with CD4 and CD8 microbeads (Miltenyi Biotec). Next, two washes were performed using CliniMACS buffer with 0.5% HSA, followed by CD4+CD8+ cell selection. The percentage of CD3+, CD4+, CD8+ and CD19+ cells in the selected population was determined by flow cytometry as quality control.Isolation of human PBMCs, CD34+ cells and gDNAPBMCs were collected from a person with X-HIGM after informed consent was provided (approved by the National Institutes of Health Institutional Review Board, protocol 94-I-0073). CD34+ cells were enriched by immunomagnetic bead selection using a CliniMACS Plus or AutoMACS instrument (Miltenyi Biotec). Human CD34+ cells were cultured in vitro using complete medium (CM) consisting of StemSpan II (StemCell Technologies) medium supplemented with 100 ng μl−1 human stem cell factor (R&D systems, 255-SC/CF), 100 ng μl−1 human thrombopoietin (R&D systems, 288-TP/CF) and 100 ng μl−1 human FLT-3 ligand (R&D systems, 308-FK/CF). Cells were seeded and maintained at a density of 0.5–1 × 106 cells per ml.For rhAmpSeq for the X-HIGM studies, gDNA was isolated from bulk edited or naive cell samples. For editing, cells were thawed and cultured for 2 days in CM before electroporation (MaxCyte) with editing reagent. gDNA was isolated at 2–5 days after electroporation as per the manufacturer’s instructions32. In MKSR studies, we used gDNA isolated from CD34+ cells for rhAmpSeq according to the previously reported protocol11.Protein production and purificationBriefly, the expression plasmids (pRha-ABE8e-NRCH, Addgene, 165417; pABE8e-protein, Addgene, 161788) were transformed into BL21Start DE3 competent cells (Thermo). ABE8-NRCH and ABE8e-NGG were expressed and purified as previously described11,37 at the Protein Production Core Facility at St. Jude. The eA3A-BE3-encoding plasmid was transformed into Rosetta 2(DE3) competent cells (MilliporeSigma) and grown in 2× tryptone yeast extract at 37 °C until reaching an optical density at 600 nm of ~2.0. Bacterial cells were induced with 0.3 mM IPTG for 20 h at 20 °C and harvested by centrifugation. Pellets were then suspended in 50 mM Tris pH 7.8, 500 mM NaCl, 10 % glycerol, 1 mM TCEP and 25 mM imidazole and lysed by passage twice through a homogenizer. The lysate was clarified by centrifugation at 20,000g for 1 h at 4 °C and the eA3A-BE3 protein was purified by Ni-NTA chromatography. The eluted protein was concentrated and treated with tobacco etch virus (TEV) protease (1 mg of lab-made TEV per 40 mg of protein) and benzonase (100 U per ml; Novagen) overnight at 4 °C. The cleaved protein was further purified by size-exclusion chromatography on a HiLoad 26/60 Superdex 200 (Amersham Biosciences) isocratically in 20 mM HEPES pH 6.8, 10% glycerol and 1 mM TCEP. Fractions containing the eA3A-BE3 were pooled and loaded onto a 5-ml SP HP column (GE) in the same buffer and eluted with a linear gradient of NaCl. Fractions were pooled and dialyzed into 20 mM HEPES pH 7.5 containing 300 mM NaCl, 10% glycerol and 1 mM TCEP. Endotoxin was removed by passage over 4 ml of Detoxigel (Pierce) and then quantitated using a toxin sensor chromogenic LAL endotoxin assay kit (GenScript). Purified proteins were concentrated using Amicon Ultra filter units (MilliporeSigma) and filtered with an ultrafree MC centrifugal filter (MilliporeSigma), followed by a 0.5 ml of 100-kDa cutoff Pierce concentrator. Protein purity was assessed on TGX stain-free 4–20% SDS–PAGE (Biorad) and quantified by rapid gold BCA assay (Thermo Fisher).Cell cultureHuman primary T cells were cultured in X-Vivo 15 medium (Lonza) supplemented with 10% human heat-inactivated serum (Fisher), 10 ng ml−1 interleukin (IL)-7 (Miltenyi) and 10 ng ml−1 IL-15 (Miltenyi) at 37 °C with 5% CO2. T cells were stimulated with MACS GMP T cell TransAct polymeric nanomatrix (Miltenyi) for 3 days according to the manufacturer’s instructions before transfection. Human primary hepatocytes were cultured in Upcyte hepatocyte high-performance medium (BioIVT) with supplement A (BioIVT) and L-glutamine (BioIVT) in plates coated with collagen type I (Sigma) at 37 °C with 5% CO2.CHANGE-seq-BE and analysisCHANGE-seq-BE was performed on gDNA isolated from human CD4+CD8+ T cells or human primary hepatocytes (BioIVT). gDNA was isolated using a PureGene tissue kit (Qiagen) and quantified by Qubit fluorimetry (Invitrogen). Purified gDNA was tagmented with a custom Tn5 transposome, gap-repaired with HiFi HotStart Uracil+ ready mix (Kapa) and Taq DNA ligase (New England Biolabs (NEB)) and treated with a mixture of USER enzyme and T4 polynucleotide kinase (NEB). DNA was circularized at a concentration of 5 ng µl−1 with T4 DNA ligase (NEB) and treated with a cocktail of exonucleases, lambda exonuclease (NEB), exonuclease I (NEB), plasmid-safe ATP-dependent DNase (Lucigen) and exonuclease III (NEB), to enzymatically degrade remaining linear DNA molecules, followed by dephosphorylation with Quick CIP (NEB). gRNAs were refolded before ABE8e:gRNA or CBE:gRNA complexing at a ratio of 1:3 to ensure full complexation of RNP. ABE in vitro deamination reactions were performed in a 50-µl volume with deamination buffer (50 mM Tris-HCl pH 8.0, 25 mM KCl, 2.5 mM MgSO4, 0.1 mM EDTA, 10% glycerol, 2 mM DTT and 10 µM ZnCl2). The final concentrations of ABE and gRNAs were 300 nM and 900 nM (sequences are listed in Supplementary Table 9), along with 125 ng of circularized DNA. In the case of in vitro deamination with CBEs, NEBuffer r3.1 was used in a 50-µl reaction with final concentrations of 300 nM CBE and 900 nM gRNA, along with 125 ng of circularized DNA. ABE8e-deaminated DNA products were treated with proteinase K (NEB), followed by endonuclease V (NEB) treatment. CBE-deaminated DNA products were treated with proteinase K (NEB), followed by USER and T4-PNK (NEB) treatment. Linearized DNA fragments were end-repaired with Klenow fragment (3′→5′ exo-) (NEB), dA-tailed, ligated with a hairpin adaptor (NEB), treated with USER enzyme and amplified by PCR using HiFi HotStart Uracil+ ready mix (Kapa/Roche). Completed libraries were quantified by qPCR using the Kapa library quantification kit (Kapa Biosystems) and sequenced with 151-bp paired-end reads on an Illumina NextSeq instrument. A detailed user protocol for CHANGE-seq-BE is provided (Supplementary Protocol). For data analyses, adaptor trimming was performed using cutadapt (version 1.18) with parameters of ‘--overlap 40 -e 0.15’ to remove the Tn5 adaptor (CTGTCTCTTATACACATCTACGTAGATGTGTATAAGAGACAG). Then, paired-end reads were mapped using bwa mem with default parameters. Reads with zero mapping quality were filtered out (for example, requiring MAPQ ≥ 1). CHANGE-seq-BE paired-end reads should be in an outward orientation with some degree of self-overlapping bases. For ABE, assuming an editing window from position 1 to position 10, we used min_overlap = 5 and max_overlap = 15 because the deamination event occurred at the +2 position. Similarly, for CBE, we used min_overlap = 6 and max_overlap = 16 because the deamination event occurred at the +1 position. Reads were filtered on the basis of the above criteria. The start positions of the valid reads were then tabulated and genomic intervals enriched in nuclease-treated samples were identified. The interval and 30 bp of flanking reference sequence on either side were searched for potential base editor off-target sites with up to six mismatches, one insertion or one deletion. The alignment distance was the sum of number of mismatches, where insertions and deletions were counted as the equivalent of three mismatches. Off-target sites with alignment distance > 6 were further selected with read count ≥ 20. For off-target sites with alignment distance ≤ 6, no read count filter was applied except for CBE, where read_count_cutoff = 6 was used. Deamination events were A-to-G conversions if the off-target strand was forward or C-to-T conversions if the off-target strand was reverse. Deamination-containing reads were counted if the deamination events occurred close to +2 of the read start position (allowing for ±2 bp of flanking). The percentage of deamination was calculated as the ratio between number of deamination-containing reads and the total number of valid reads assigned to the off-target site. CHANGE-seq-BE data analyses were performed using open-source software (https://github.com/tsailabSJ/changeseq/tree/BE).CHANGE-seqCHANGE-seq was performed as previously described20. Briefly, purified gDNA was tagmented with a custom Tn5 transposome, followed by gap repair with Kapa HiFi HotStart Uracil+ DNA polymerase (KAPA Biosystems) and Taq DNA ligase (NEB). Gap-repaired tagmented DNA was treated with USER enzyme (NEB) and T4 polynucleotide kinase (NEB). Intramolecular circularization of the DNA was performed with T4 DNA ligase (NEB) and residual linear DNA was degraded by a cocktail of exonucleases containing plasmid-safe ATP-dependent DNase (Lucigen), lambda exonuclease (NEB) and exonuclease I (NEB). In vitro cleavage reactions were performed with 125 ng of exonuclease-treated circularized DNA, 90 nM SpCas9 protein (NEB), NEB buffer 3.1 (NEB) and 270 nM gRNA in a 50-μl volume. Cleaved products were dA-tailed, ligated with a hairpin adaptor (NEB), treated with USER enzyme (NEB) and amplified by PCR with barcoded universal primers NEBNext Multiplex Oligos for Illumina (NEB), using Kapa HiFi Polymerase (KAPA Biosystems). Libraries were quantified by qPCR (KAPA Biosystems) and sequenced with 151-bp paired-end reads on an Illumina NextSeq instrument. CHANGE-seq-BE data analyses were performed using open-source software (https://github.com/tsailabSJ/changeseq).Digenome-seq and analysisDigenome-seq performed as previously described for ABE and CBEs38. RNP complexation of ABEs and CBEs to gRNA occurred at a ratio of 1:3 with final concentrations of 150 nM ABEs or CBEs and 450 nM gRNAs (refolded before complexation), followed by incubation at 37 °C for 10 min. A total of 5 μg of gDNA was treated with ABE8e-NGG-complexed and eA3A-BE3-complexed RNPs for in vitro deamination with respective buffers at 37 °C for 8 h. Monarch RNAse A (NEB) was added before incubating at 37 °C for 15 min, followed by proteinase K (NEB) treatment, incubation at 37 °C for 15 min, purification with 1× volume of AMPure XP beads and elution in 1× Tris–EDTA (Integrated DNA Technologies (IDT)). Next, deaminated gDNA was treated with endonuclease V for ABE-treated samples and USER for CBE-treated samples with respective conditions. A total of 3 μg of purified in vitro ABE-deaminated DNA was mixed with 4 μl of endonuclease V (10 U per μl) and 20 μl of NEB buffer 4 (10×) in a final volume of 200 μl with nuclease-free water and incubated at 37 °C for 2 h. On the other hand, 2 μg of purified in vitro CBE-deaminated DNA was treated with 6 μl of USER (1 U per μl) and 10 μl of NEB cut smart buffer (10×) in a final volume of 100 μl with nuclease-free water and incubated at 37 °C for 3 h. Next, the final reaction was treated with proteinase K (NEB) at 37 °C for 15 min, followed by 1× volume of bead purification, and quantified by Qubit HS. A total of 1 μg of in vitro deaminated gDNA was fragmented to the 550–750 bp using Covaris (Life Technologies) with the following settings: duty factor, 20%; peak displayed power, 50 W; cycles per burst, 200; duration, 65 s; temperature, 20 °C. Later, sheared DNA was end-repaired and dA-tailed using Kapa HyperPrep PCR free (Kapa), ligated with adaptor stock (UDI-UMI, IDT), purified twice with 0.8× beads and eluted in 1× Tris–EDTA. Libraries were subjected to WGS to sequence with 20–30× coverage on Novaseq 6000 with 150–17–8–150 cycles. All samples were normalized to 400 million reads and analyzed using Digenome-Seq standalone software (version 1.0) with default parameters: MAPQ score ≥ 1, a minimum of five forward reads with identical 5′ ends, a minimum of five reverse reads with identical 3′ ends, a minimum read depth of 10 at each position, a minimum cleavage ratio of 0.2 and a minimum cleavage score of 2.5 (ref. 39). Editing windows and read overlap lengths were consistent with those used in CHANGE-seq-BE: 1–10-long editing window, 5–15-long read overlap for ABE and 6–16-long read overlap for CBE. Potential off-target sites containing up to six mismatches, one insertion or one deletion that overlapped the Digenome cut sites were identified as nuclease-induced candidates. Digenome-seq data analyses were performed using open-source code (http://www.rgenome.net/digenome-js/standalone).Cell transfectionTransfections of human primary T cells were performed with 2 µg of ABE8e, eA3A-BE3 or Cas9 mRNA and 1 µg of synthetic chemically modified gRNA. ABE8e in vitro transcribed mRNA was commercially synthesized by TriLink Biotechnologies. eA3A-BE3 in vitro transcribed mRNA was synthesized using cotranscriptional capping using CleanCap reagent AG from TriLink and a HiScribe T7 high-yield RNA synthesis kit from NEB according to the manufacturer’s instructions. Synthetic gRNAs ordered from Synthego (same gRNAs applied in CHANGE-seq and CHANGE-seq-BE) were used for electroporation to perform hybrid capture sequencing. mRNA:gRNA was added directly to 1 × 106 cells resuspended in 20 µl of P3 solution and nucleofected with preprogrammed pulse EO-115 in a 4D-Nucleofector system (Lonza). After nucleofection, cells were recovered in X-vivo 15 medium with 20% human heat-inactivated serum (Fisher), 10 ng ml−1 IL-7 (Miltenyi) and 10 ng ml−1 IL-15 (Miltenyi). After 5 days, cells were harvested for gDNA purification using Agencourt DNAdvance (Beckman Coulter). Transfection of human primary hepatocytes was performed using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher). Briefly, 1.5 × 105 hepatocytes were seeded in a six-well plate coated with collagen type I. After 24 h, cells were transfected with 2.5 µg of ABE8e mRNA, 2.5 µg of synthetic chemically modified gRNA (PCSK9) and 7.5 µl of Lipofectamine MessengerMAX, following the manufacturer’s recommendations. After 3 days, cells were harvested for gDNA purification using Agencourt DNAdvance (Beckman Coulter).Multiplex targeted amplicon sequencingTo determine the A-to-G conversion frequency at CHANGE-seq-BE-identified sites, on-target and off-target sites were amplified from gDNA from edited cells or unedited controls using the rhAmpSeq system (IDT), in triplicate, with primers listed in Supplementary Table 2. Note that, for the validation of the newly CHANGE-seq-BE-detected sites for MKSR targeting, we used the same gDNA of CD34+ cells treated with ABE8e-NRCH:HBBS-gRNA mRNA described in our previous study. Sequencing libraries were generated according to the manufacturer’s instructions. Completed libraries were quantified by qPCR using the Kapa library quantification kit (Kapa Biosystems) and sequenced with 151-bp paired-end reads on an Illumina MiSeq or MiniSeq instrument.GUIDE-seq-2 and analysisGUIDE-seq-2 was performed as previously reported29. Briefly, 100 µM assembled double-stranded oligodeoxynucleotide (dsODN) tag was prepared by annealing forward and reverse dsODN oligos in 1x STE buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA), heated at 95 °C for 5 min and gradually cooled down to 25 °C in a thermocycler. For RNP, Cas9-3xNLS (15 µM), gRNA (45 µM), 1 µl assembled dsODN (100 µM) were used to nucleofect 0.6 × 106 human primary T cells. For mRNA, 1 × 106 cells were nucleofected with Cas9 (2 µg), gRNA (1 µg) and 1 µl assembled dsODN (100 µM), and cells were recovered in supplemented X-vivo 15 media and harvested after 3 days (RNP) and 5 days (mRNA) of nucleofection. gDNA from edited and control cells containing the dsODN tag was isolated using the DNAdvance kit (Beckman Coulter) according to the manufacturer’s instructions. Purified gDNA was quantified by Qubit BR and 100 ng of gDNA was tagmented to an average length of 600 bp with a Tn5 transposome, purified from pTXB1-Tn5 (Addgene, 60240), assembled with oligos containing a 10-nt unique molecular identifier and then purified using solid-phase reverse immobilization (SPRI) guanidine magnetic beads. Tagmented gDNA was PCR-amplified using dsODN sense-specific and antisense-specific primers in separate reactions for each sample for target enrichment, which were further purified with a limited number of SPRI magnetic beads for one-step purification and normalization. Libraries for the replicates were pooled and size selected for 250–450 bp by Lightbench (Yourgene Health), quantified by a qPCR library quantification kit (TakaraBio) and sequenced with 146-bp paired-end reads on an Illumina NextSeq 2000. GUIDE-seq-2 data analyses were performed using open-source software (https://github.com/tsailabSJ/guideseq).Hybrid capture sequencingFirst, 50 ng of gDNA per sample was used for enzymatic fragmentation at 4 °C (∞), 37 °C (20 min), 65 °C (30 min) and 4 °C (∞) using the Twist library preparation EF kit 2.0. Twist UMI adaptors were ligated to the dA-tailed DNA fragments, incubated at 20 °C for 15 min and later purified with DNA purification beads. Adaptor-ligated gDNA libraries were amplified with Twist UDI primers with the following PCR conditions: 98 °C for 45 s (denaturation), followed by eight cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s (annealing) with a final extension at 72 °C for 1 min, before holding at 4 °C. PCR-indexed gDNA libraries were then purified with beads, quality-controlled on an Agilent D1000 Tapestation with the expected size of library ~350–425 bp and quantified by Qubit BR. The indexed samples were pooled at a concentration of 250 ng and the total mass of the each pool was ~2,000 ng. The eight multiplexed libraries were dried with a speed vacuum and hybridized with respective ABE and CBE panels of custom probes (Twist) at 70 °C for 17 h (lid at 85 °C). Next, hybridized targets were captured with streptavidin beads and PCR was used to amplify postcaptured products according to the manufacturer’s instructions. The ABE panel PCR conditions were as follows: 98 °C for 45 s (denaturation), followed by nine cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s (annealing) with a final extension at 72 °C for 1 min (final extension), before holding at 4 °C. The CBE panel PCR conditions were as follows: 98 °C for 45 s (denaturation), followed by 11 cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s (annealing) with a final extension at 72 °C for 1 min, before holding at 4 °C. Postcaptured PCR products were purified using DNA purification beads and enriched libraries were quantified by Qubit HS. The library size was determined on an Agilent D1000 HS Tapestation to be ~350–450 bp. CBE and ABE libraries were pooled and loaded separately on NextSeq 2000 and NovaSeq X paired-end sequencing with 146–15–15–146 cycles according to the manufacturer’s instructions. Paired-end sequencing reads were trimmed using cutadapt with ‘-a AGATCGGAAGAGC -A AGATCGGAAGAGC’. Then, FLASH was used to merge R1 and R2 reads. Unmerged reads were treated as individual molecules. Merged and unmerged reads R1/R2 were concatenated. Unique molecular identifier deduplication was performed using open-source software (https://github.com/aryeelab/umi). Unique molecular identifier deduplicated reads were mapped to hg38 using bwa mem with default parameters. Lastly, CRISPRessoWGS was used to quantify indel frequency and base-editing frequency. A description of the analysis is available online (https://hemtools.readthedocs.io/en/latest/content/NGS_pipelines/hybrid_capture.html). The source code is available from GitHub (https://github.com/YichaoOU/HemTools/blob/master/share/lsf/hybrid_capture.lsf).Targeted sequencingTo determine the A-to-G conversion frequency at intended and unintended on-target sites, primary human T cells were nucleofected with ABE8e mRNA and CBLB or CIITA gRNAs. On-target sites (CBLB, CIITA, B2M, CD7 or PDCD1) were amplified from T cell gDNA using 2× Phusion HotStart flex master mix (NEB) (primers ordered from IDT listed in Supplementary Table 9) and 100 ng of gDNA as the input for each PCR. PCR products were purified with SPRI magnetic beads and normalized. Then, 10 ng of each amplicon were used for the second PCR to add complete Illumina adaptors and indices. Completed libraries were quantified by qPCR using the Kapa library quantification kit (Kapa Biosystems) and sequenced with 151-bp paired-end reads on an Illumina MiSeq or MiniSeq instrument.gRNA synthesis, sequencing and analysisSynthetic gRNAs ordered from BioSpring, Synthego and IDT were used for the experiments. Two sets of synthetic gRNAs from BioSpring were tested. Both sets were HPLC-purified and one set used an HPLC column clearing-in-place procedure between gRNA purifications. Synthetic gRNAs from Synthego and IDT were purified using a standard desalting method. These gRNAs were chemically modified with 2′-O-methyl analogs and 3′ phosphorothiate internucleotide linkages at the first three 5′ and 3′ terminal residues (Supplementary Table 9). To generate gRNA-seq libraries, we repurposed the SMARTer smRNA-seq kit (Takara). First, cDNA synthesis was performed with 1–2 ng of gRNA template using 1 μl of custom-tailed scaffold specific primer (10 μM) 5′ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCCACTTTTTCAAGTTG 3′. The reaction was incubated at 72 °C for 3 min and immediately placed on ice for 2 min to anneal a custom-tailed scaffold primer at the 3′ end of the scaffold gRNA. cDNA synthesis, library enrichment and sequencing were performed according to the manufacturer’s instructions, as previously reported28. gRNA sequencing data analyses were performed using open-source software (https://github.com/tsailabSJ/gRNA_sequencing).Quantification analysis of base-editing activity, annotation and statistical analysesThe base-editing frequency for each position in the protospacer was quantified using CRISPRessoPooled (version 2.0.41). For the statistical analysis shown in dot blots between mRNA treatments and control samples (n = 2 or 3), a χ2 test was performed, followed by false discovery rate (FDR) calculation using the Benjamini–Hochberg method. The reported off-target sites with difference ≥ 0.5% for at least one treatment and with FDR ≤ 0.05 were determined as significant. Custom code was used to conduct off-target quantification and the statistical analysis method is available from GitHub (https://github.com/tsailabSJ/MKSR_off_targets). The genomic features of all nominated and bona fide off-target sites were annotated using HOMER (version 4.10)40. Standard statistical analyses were performed using GraphPad Prism version 10.5.0. The statistical t-tests, exact tests, methods, P values and numbers of biological replicates are provided in the figure legends. Venn diagrams and Manhattan plots were created using R version 4.5.1.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.Data availabilityAll high-throughput sequencing data generated in this study (CHANGE-seq, CHANGE-seq-BE, Digenome-seq, GUIDE-seq, rhAmpSeq, hybrid capture sequencing and gRNA sequencing) were deposited to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE298535. All additional data relevant to the main results of this study are available within the article and Supplementary Information.Code availabilitySource code for all methods and sequencing data analyses are available on GitHub: CHANGE-seq (https://github.com/tsailabSJ/changeseq), CHANGE-seq-BE (https://github.com/tsailabSJ/changeseq/tree/BE), gRNA sequencing (https://github.com/tsailabSJ/gRNA_sequencing), GUIDE-seq-2 (https://github.com/tsailabSJ/guideseq), hybrid capture (https://github.com/YichaoOU/HemTools/blob/master/share/lsf/hybrid_capture.lsf), targeted sequencing (https://github.com/tsailabSJ/count_indels_integrations) and rhAmpSeq and χ2 test (https://github.com/tsailabSJ/MKSR_off_targets).ReferencesKomor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).Article CAS PubMed PubMed Central Google Scholar Gaudelli, N. M. et al. 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Heath from the St. Jude Protein Production Core Facility for recombinant Tn5 and recombinant ABE8e and eA3A-BE3 expression and purification and J. Yen and M. Weiss for ABE8e-NRCH-HBB-edited CD34+ cell gDNA. This work was supported by the St. Jude Children’s Research Hospital, the American Lebanese Syrian Associated Charities, the National Institutes of Allergy and Infectious Diseases (U01AI176470 and U01AI176471, to S.Q.T.), the National Heart Lung and Blood Institute (U01HL163983, to S.Q.T.), the National Cancer Institute (75N91019D00024 to X.W.), the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Z01-Al-00644, Z01-AI-00645, and Z01-Al-00988 to S.S.D.R.), the St. Jude Collaborative Research Consortium on Novel Gene Therapies for Sickle Cell Disease, the St. Jude PARADIGM blue sky project and the Doris Duke Charitable Foundation (2020154).Author informationAuthor notesElizabeth UrbinaPresent address: Vanderbilt University, Nashville, TN, USAGaHyun LeePresent address: Northwestern University Feinberg School of Medicine, Chicago, IL, USAThese authors contributed equally: Cicera R. Lazzarotto, Varun Katta.Authors and AffiliationsDepartment of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USACicera R. Lazzarotto, Varun Katta, Yichao Li, Garret Manquen, Rachael K. Wood, Jacqueline Chyr, Elizabeth Urbina, Azusa Matsubara, GaHyun Lee & Shengdar Q. TsaiCancer Research Technology Program, Frederick National Laboratory of Cancer Research, Frederick, MD, USAXiaolin WuGenetic Immunotherapy Section, LCIM, NIAID, National Institutes of Health, Bethesda, MD, USASuk See De RavinAuthorsCicera R. LazzarottoView author publicationsSearch author on:PubMed Google ScholarVarun KattaView author publicationsSearch author on:PubMed Google ScholarYichao LiView author publicationsSearch author on:PubMed Google ScholarGarret ManquenView author publicationsSearch author on:PubMed Google ScholarRachael K. WoodView author publicationsSearch author on:PubMed Google ScholarJacqueline ChyrView author publicationsSearch author on:PubMed Google ScholarElizabeth UrbinaView author publicationsSearch author on:PubMed Google ScholarAzusa MatsubaraView author publicationsSearch author on:PubMed Google ScholarGaHyun LeeView author publicationsSearch author on:PubMed Google ScholarXiaolin WuView author publicationsSearch author on:PubMed Google ScholarSuk See De RavinView author publicationsSearch author on:PubMed Google ScholarShengdar Q. TsaiView author publicationsSearch author on:PubMed Google ScholarContributionsC.R.L., V.K. and S.Q.T conceptualized and designed the study. S.Q.T. supervised the research. C.R.L., V.K., G.M., R.K.W., E.U., A.M., G.L. and X.W. performed the experiments. S.S.D.R. provided conceptual assistance. Y.L. and J.C. performed the computational analysis. C.R.L., V.K. and S.Q.T. wrote the paper with input from all authors.Corresponding authorCorrespondence to Shengdar Q. Tsai.Ethics declarationsCompeting interestsC.R.L. and S.Q.T. are inventors on a patent covering CHANGE-seq. S.Q.T. is an inventor on patents covering CIRCLE-seq and GUIDE-seq. S.Q.T. is a member of the scientific advisory board of Prime Medicine and Ensoma. The other authors declare no competing interests.Peer reviewPeer review informationNature Biotechnology thanks Samuele Ferrari, Luigi Naldini and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 CHANGE-seq-BE optimization for adenine base editor (ABE) and cytosine base editor (CBE).a, Bar plot showing the percentage of ABE8e-NRCH:HBBS-gRNA previously validated off-target sites detected by CHANGE-seq-BE on a subset of enzymatic conditions systematically evaluated during the method development. b, In vitro cleavage profile of RNF2 amplicon treated independently with AnBE4max, A3A-BE3, eA3A-BE3, Td-CGBE, and Td-CBEmax proteins respectively in a single experiment to test biochemical activity.Extended Data Fig. 2 CHANGE-seq-BE detects new off-target sites for ABE8e-NRCH:HBBS-gRNA.a, Scatterplot showing correlation of CHANGE-seq-BE read counts using ABE8e-NRCH and CIRCLE-seq using cognate Cas9-NRCH, colored by the number of adenines in the protospacer positions. b, Venn diagram depicting the overlap of off-target sites detected by CHANGE-seq-BE ABE8e-NRCH and CIRCLE-seq Cas9-NRCH for the HBBS-gRNA target. c, Pie chart of genomic annotations of all validated ABE off-target sites for the ABE8e-NRCH:HBBS-gRNA target site.Extended Data Fig. 3 CHANGE-seq-BE identifies bona fide off-targets for ABE targeting PCSK9 and CD7.a, Schematic illustrating CHANGE-seq-BE off-target nomination in primary human hepatocytes targeting PCSK9. b, Manhattan plot showing the genome-wide distribution of CHANGE-seq-BE detected sites (on-target site indicated with pink arrow). c, Bona fide CHANGE-seq-BE off-targets in primary hepatocytes validated by hybrid capture sequencing with read counts to the left of off-target sites. Mismatches to intended target site indicated with colored nucleotides, matches indicated with dots. Dot plot showing the percentage of sequencing reads containing A-to-G mutations within the protospacer positions 1-10 at off-target sites in genomic DNA samples from primary human hepatocytes for PCSK9 treated with ABE8e-NGG mRNA, or untreated controls (n = 3). d, Remaining validated off-targets confirmed by hybrid capture sequencing for CD7 target. One statistically significant off-target (OT 16) with less than 1000 hybrid capture reads annotated with †. e, Predicted genomic features of CHANGE-seq-BE and Digenome-seq (TTS - transcription termination site, TSS - transcription start site, and UTR - untranslated region). Panel a created with BioRender.com.Extended Data Fig. 4 gRNA sequencing identifies potential gRNA contaminants in CBLB and CIITA gRNAs.a, Bar plot showing the percentage of gRNA sequencing reads mapping to target gRNAs for the three synthetic gRNAs manufacturers. Striped bars indicate gRNA contaminants.Extended Data Fig. 5 Correlation profiles of CHANGE-seq and CHANGE-seq-BE for ABE and CBE targets.a, Scatterplot showing read counts correlation for two independent CHANGE-seq (Cas9) technical replicates. b, Scatterplot showing read counts correlation for two independent CHANGE-seq-BE technical replicates for ABE8e and eA3A-BE3.Extended Data Fig. 6 GUIDE-seq-2 identifies off-targets for Cas9 delivery as mRNA and RNP.a, Bar plot showing the on-target editing activity of Cas9-mRNA with and without dsODN. Shaded region indicates the percentage of dsODN tag integration. b, Alignment plots for the off-target sites identified by GUIDE-seq-2 using Cas9 as RNP vs mRNA for five ABE targets (n = 3). Percentage of off-target reads normalized relative to on-target reads shown column-wise for Cas9 as RNP and mRNA.Extended Data Fig. 7 CHANGE-seq-BE as an IND-enabling assay to characterize off-target activity of ABE8e (HF1) targeting CD40L mutation to treat X-HIGM patient.a, Manhattan plot of CHANGE-seq-BE detected on- and off- targets across the genome for CD40L target corrected by ABE8e (HF1). b, Scatterplot showing read counts and correlation between two independent experimental replicates. c, Genomic features for on- and off-target sites identified by CHANGE-seq-BE (TTS - transcription termination site, TSS - transcription start site). d, Validation of CHANGE-seq-BE nominated off-targets by multiplex targeted sequencing (rhAmpSeq, IDT). The x-axis represents chromosome location and y-axis represents percentage of A-to-G base editing by ABE8e (HF1).Supplementary informationSupplementary InformationSupplementary Notes 1 and 2, Protocol and References.Reporting SummarySupplementary TablesThis file contains Supplementary Tables 1–9. Supplementary Table 1 contains a complete list of CHANGE-seq-BE-detected sites for MKSR. Supplementary Table 2 contains a list of rhAmpSeq-validated sites for MKSR. Supplementary Table 3 contains a complete list of sites detected by CHANGE-seq-BE, Digenome-seq and CHANGE-seq for ABEs targeting B2M, CBLB, CD7, CIITA, PDCD1 and PCSK9. Supplementary Table 4 contains a list of hybrid-capture-validated sites for sites nominated by CHANGE-seq-BE, Digenome-seq and CHANGE-seq as ABE targets. Supplementary Table 5 contains a list of additional hybrid-capture-validated sites with relaxed CHANGE-seq-BE parameters for ABE targets. Supplementary Table 6 contains a complete list of sites detected by CHANGE-seq-BE and Digenome-seq for CBE targeting B2M, CBLB, CD7, CIITA and PDCD1. Supplementary Table 7 contains a list of hybrid-capture-validated sites for sites nominated by CHANGE-seq-BE and Digenome-seq as CBE targets. Supplementary Table 8 contains a list of missed CHANGE-seq-BE sites in ABE validation. Supplementary Table 9 contains gRNA sequences for ABE and CBE targets and the next-generation sequencing primer list.Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. 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