Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope

Domain: chemistry
Published: 2026-02-19
Editorial outcome: major_revision

Abstract
The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.

Introduction
Originally discovered in bacteria and archaea as an adaptive immune mechanism, the CRISPR-Cas system has since evolved and been adapted into revolutionary tools for genome editing1–3. Among the six types of the CRISPR systems belonging to two classes, types II, V, and VI are composed of a single effector nuclease that can be flexibly guided by engineered single guide RNAs (sgRNAs), and they attract more attention for multiplex applications4–6. Compared to the widely used type II Cas9 system, which primarily recognizes G-rich protospacer adjacent motifs (PAMs), the type V CRISPR-Cas12 system relies on T-rich or a variety of PAMs with high specificity7,8. Although SpCas9 has been engineered for unconstrained genome targeting, its large size still limits its application due to in vivo delivery challenges9–11. In recent years, the identification of Cas12 family flourished and at least 14 distinct subtypes have been discovered with a wider range of protein sizes from 400 to 1500 amino acids (aa)12,13, including recently reported highly compact Cas12 systems (less than 700 aa), such as Cas12m (~600 aa), Cas12n (400–700 aa), and Cas12f (400–700 aa)14–16. These miniature Cas enzymes have attracted a lot of attention since their small size is appropriate for packaging into adenovirus-associated virus (AAV) vectors, which are the most successful delivery system for in vivo gene therapy17–20, despite having a limited packaging capacity of ~4.7 kb21. However, most of these compact Cas12 proteins demonstrate only modest or even infrequent editing activity in their natural state in mammalian cells.

Un1Cas12f1, the first characterized member of the Cas12f family, was originally identified as a programmable nuclease targeting single-stranded DNA (ssDNA) and subsequently demonstrated to cleave double-stranded DNA16,22. Un1Cas12f1 shows great promise for genome editing due to its compact size and high specificity23. Later, its cleavage activity has been improved through sgRNA scaffold optimization and protein engineering24,25. Following the success of Un1Cas12f1 optimization, several other members of the Cas12f family, which recognize various PAM sequences, have been characterized. These include AsCas12f1 (PAM: NTTR), SpaCas12f1 (PAM: NTTY), RhCas12f1 (PAM: NCCD), CnCas12f (PAM: NCCD), and OsCas12f1 (PAM: YTTH)22,26–29. Leveraging their small size for efficient delivery, some of these Cas12f members have been adapted to develop base editors and transcriptional regulatory tools, potentially for in vivo gene therapy24,30–33. Although both the Cas12f enzymes and guide RNAs have undergone engineering to improve their editing efficiency, their activity remains suboptimal. Additionally, the targeting scope of engineered Cas12f variants is limited by their complex PAM sequence requirements. Therefore, there is an urgent need for highly efficient miniature Cas proteins with broad PAM compatibility to unlock their full potential across diverse genome editing applications.

To expand the targeting scope, we establish bacterial positive library selection and endogenous target screening to develop evoCas12f, a variant containing five amino acid substitutions that exhibits a broader targeting scope and higher editing efficiency. evoCas12f expands the PAM recognition range from the original TTTR to a more permissive NTNR or NYTR spectrum. Compared to wild-type Un1Cas12f1, evoCas12f demonstrates an average 12.5-fold increase in cleavage efficiency across all permissible PAMs, achieving editing rates of up to 91% in mammalian cells and generating homozygous mouse model in F0 generation at non-TTTR PAM targets. Notably, evoCas12f shows superior editing activity compared to previously engineered Cas12f variants, such as enAsCas12a and other Cas12f family members, under similar PAM contexts. Furthermore, we employ the strategy of using engineered sgRNA to recruit effector proteins to develop adenine base editors (ABEs), cytosine base editors (CBEs), and transcriptional activators based on evoCas12f. Collectively, these findings demonstrate that evoCas12f is a compact, highly efficient, and versatile genome-editing platform with a broadened targeting scope.

Results
Relaxing the PAM recognition profile using a point mutation library

Mutations within the PAM-interacting region are likely to alter the PAM recognition spectrum of a Cas effector9–11,34–40. To explore this, we constructed a plasmid library containing single-point saturation mutations across the PAM-interacting regions (K129-S168, K196-I207, Q242-K249) of Un1Cas12f1 whose expression was induced by IPTG. A previously reported bacterial positive selection system41 was leveraged for the screening (Fig. 1a). In this system, a screening plasmid containing a target with a specific PAM sequence followed by an arabinose-inducible ccdb suicide gene was first introduced into E. coli. During the screening process, the mutant library was electroporated into the competent E. coli cells, induced by IPTG, and plated on LB agar containing arabinose. The survival of E. coli depended on the ability of the Cas12f variant to recognize the PAM and cleave the target sequence, thus preventing the expression of the suicide gene. To validate this system, we electroporated WT-Un1Cas12f1 into cells containing the TTTG-PAM selection plasmid or negative control plasmid (no target sequence is included). As expected, the growth of E. coli containing the negative control plasmid was completely inhibited after arabinose induction. Cleavage of the selection plasmid by WT-Un1Cas12f1 restored the growth of competent cells (Supplementary Fig. 1a and b). When the TTTG-PAM was replaced with non-canonical PAMs, E. coli survival decreased very dramatically, confirming that WT-Un1Cas12f1 exhibits very minimal activity on non-TTTG PAMs in bacteria (Supplementary Fig. 1c).

Next, we electroporated the Un1Cas12f1 library into four batches of competent cells, each harboring one of the NTTR-PAMs. Colonies that grew on LB agar containing arabinose were harvested the following day and analyzed by Next-Generation Sequencing (NGS). We cloned 38 Un1Cas12f1 variants that showed over 100-fold enrichment into eukaryotic-expression plasmids (Fig. 1b). Their editing efficiencies in HEK293T cells were evaluated at three endogenous sites of each NTTR PAM. We discovered three point mutations (D143S, L152S and Q244R) that exhibited the highest editing efficiencies at (T/C/A/G)TTR PAM targets, respectively (Fig. 1c and Supplementary Data 1). To develop mutants with broader PAM compatibility, we integrated D143S, L152S and Q244R to develop Cas12f-SSR. Then we tested 14 endogenous targets with NTTR PAMs to evaluate its performance. The results demonstrated that Cas12f-SSR increased its average efficiency from 5.0% to 24.2% on VTTR PAMs, and an average 1.6-fold improvement at TTTR sites compared to WT-Un1Cas12f1 (Fig. 1d).

To further relax the PAM requirement at the second position of the TTTR PAM, we conducted a second round of screening against TVTR PAMs (V=A/C/G) through the same methods used above. We discovered the N133S variant exhibited increased activity at two of three endogenous targets with TATR PAMs, while E151V outperformed other variants on TGTR PAMs (Fig. 1e, f). Then we incorporated N133S and E151V into Cas12f-SSR and resulted in the Cas12f-SSRSV (hereafter referred as evoCas12f). Next, using 24 endogenous sites with NTTR or TNTR PAM in HEK293T cells, we tried to evaluate whether evoCas12f had expanded PAM recognition. Notably, evoCas12f showed cleavage efficiencies ranged from 14.79% to 71.41% across all these targets, with a broader PAM compatibility compared to WT-Un1Cas12f1 (Fig. 1g).

Arginine (Arg) substitutions are often used to strengthen DNA–protein interactions. Motivated by the benefit of Q244R, we introduced individual Arg substitutions at N133, D143, L152, and E151 and also evaluated combinatorial variants. However, the results indicated that D143S, L152S, N133S, and E151V each had generally higher editing efficiency than their to-arginine counterparts at the corresponding PAM endogenous sites. (Supplementary Fig. 2a–d) Moreover, the five-Arg construct (N133R/D143R/E151R/L152R/Q244R) exhibited consistently lower activity on NTTR and TNTR PAM sites (Supplementary Fig. 2e). To investigate potential mechanisms, we analyzed the structure of Un1Cas12f1-sgRNA-dsDNA (PDB: 7C7L) and performed corresponding in silico point mutations. Our analysis revealed that Q244R, extends deeper into the DNA minor groove, forming hydrophilic interactions and salt bridges with phosphate groups to enhance editing efficacy and broaden PAM compatibility via non-specific affinity (Supplementary Fig. 3). Among the non-arginine mutations, N133S may promote DNA strand separation near the cleavage site; D143S and E151V may function by neutralizing acidic residues to alleviate electrostatic repulsion near the PAM-interacting DNA backbone; while L152S may improve steric compatibility with surrounding polar residues to harmonize the local environment (Supplementary Fig. 3; see Discussion).

Characterization of the activity and PAMs of evoCas12f

To further characterize the activity of evoCas12f on more relaxed PAM targets, we tested 40 endogenous sites containing NNTR PAMs in HEK293T cells (Supplementary Data 1). The editing efficiencies of WT-Un1Cas12f1 were below 5% at most NVTR sites and less than 10% at the VTTR PAM (V=A/G/C) sites (Fig. 2a and Supplementary Fig. 4). In contrast, evoCas12f exhibited higher editing efficiency across all NNTR sites, with an average increase of 15.4-fold (Fig. 2a, d). Notably, WT-Un1Cas12f1 displayed extremely low efficiency at GCTG-2 site (0.2%), while evoCas12f achieved 19.93% indel efficiency. Statistically, evoCas12f exhibits a 20-fold increase in activity on NVTR PAM targets (Fig. 2a).

To validate the PAM preference of evoCas12f statistically, we employed an HEK293T cell line harboring a GFP reporter system42. This system includes a GFP gene preceded by a target sequence with a N5 PAM after the ATG start codon, resulting in a frameshift mutation. Cleavage by the Cas12f protein introduces indels in the protospacer, potentially restoring the GFP reading frame (Supplementary Fig. 5). As expected, WT-Un1Cas12f1 exhibited a TTTR PAM, while evoCas12f displayed a more relaxed NTNR PAM, maintaining a slight bias toward ‘T’ at position 2 (Fig. 2b). It is worth noting that the second thymidine in the PAM was not strictly required, as demonstrated in Fig. 2a Supplementary Fig. 4.

To address evoCas12f’s mild preference for T at PAM position 2, structural analysis identified Y146 and Q197 as contacting the second T of the PAM (Supplementary Fig. 6a). We conducted single amino acid saturation mutagenesis at each residue and tested editing at 6 VVTR sites. The results showed that any substitution at Y146 abolished its activity, whereas Q197K increased its activity at 4/6 VTTR PAM sites (Supplementary Fig. 6b). We then evaluated Q197K at 40 NNTR endogenous loci and found that Q197K boosts activity on NCTR PAMs, has minimal effect on NRTR, and reduces activity on NTTR to near WT Un1Cas12f1 levels (Supplementary Fig. 6c). Collectively, Q197K does not expand evoCas12f’s overall NNTR targeting range but shifts the preference of the 2nd position from T to C. Thus, evoCas12f (Q197K) is best considered a specialized variant for NCTR targets.

To further evaluate the activity of evoCas12f on NTNR PAM targets, we tested additional 40 endogenous sites. For the ATNR, CTNR, and GTNR PAM targets, evoCas12f exhibited comparable average editing efficiencies (22.42%, 20.69%, and 21.24% respectively), representing an ~11-fold increase compared to the WT control (Fig. 2c). Among all endogenous targets with NNTR and NTNR PAM tested, evoCas12f demonstrated a 12.5-fold average increase in editing efficiency compared to WT-Un1Cas12f1 (Fig. 2d). Based on results of GFP reporter assay and endogenous cleavage experiments, we defined evoCas12f’s PAM specificity as 5’-NTNR-3’ and 5’-NYTR-3’ (Y=C/T), although some of the NATR and NGTR sites also showed efficient (>20%) editing (Fig. 2b, Supplementary Fig. 7). We further analyzed the distribution of PAM sequences suitable for evoCas12f or WT-Un1Cas12f1 in the human genome. evoCas12f increased the potential cleavage sites by 13.3-fold, reducing the median distance between adjacent cleavage sites to 2 bp, whereas WT-Un1Cas12f1 primarily targeted sites spaced >30 bp apart (Fig. 2e).

Comparing editing activity of evoCas12f with other engineered Cas12 proteins

Next, we compared evoCas12f’s cleavage efficiency with other engineered Cas12 proteins. Compared to enAsCas12a (1308aa), which recognizes expanded PAMs from TTTV to TTYN, VTTN, and TRTV sequence, evoCas12f exhibited comparable cleavage efficiency (69.0% VS 70.7%), indicating its activity was on a level with the large size Cas12 variant (Fig. 2f, g). We further evaluated evoCas12f against other miniature Cas12 members including CasMINI24, CWCas12f30 variants, AsCas12f variants43, enOsCas12f127 and enRhCas12f127. As shown in Supplementary Fig. 8, across 47 endogenous targets spanning five compact Cas12f families, evoCas12f displayed generally superior or comparable activity. Notably, evoCas12f achieved higher cleavage activity at all matched targets than other engineered Cas12f variants recognizing T-rich PAMs. It also showed superior activity at 4 of 6 target sites compared to enRhCas12f1, which binds the 5′-CCD (D=A/G/T), a non-T-rich PAM (Supplementary Fig. 8e). Compared with other engineered Cas12 variants, evoCas12f combines a compact structure, broadened targeting range, and enhanced cleavage activity, highlighting its potential for applications.

Generating Un1Cas12f1-ABE via RNA-protein recruitment modules

The compact size of Un1Cas12f1 makes it an ideal basal module for CRISPR-derived tools, such as base editors and epigenome regulators. Although several Cas12f related compact base editors and transcriptional regulatory tools have been reported24,30–33,43, their activity, editing window and targeting flexibility remain suboptimal. In our preliminary experiments, fusing TadA8e directly to the N- or C-terminus of wild typed Un1Cas12f1 yielded only limited editing efficiency (Supplementary Fig. 9). We reasoned that Cas12f likely functions through dimerization, potentially obstructing TadA8e from accessing the substrate as it also functions as a dimer.

To address this limitation, we adopted an alternative strategy by incorporating an MS2 aptamer into the sgRNA stem loop to recruit the MCP-TadA8e fusion protein (Fig. 3a). Four constructs engineered with TadA8e at varying positions relative to MCP and dUn1Cas12f1 were tested. All constructs induced A-to-G conversions within the target site (Fig. 3b, c). Among them, construct C1 showed the highest editing efficiency at the A3 position of the two sites tested (Fig. 3c). Meanwhile, we screened optimal insertion sites for the MS2 aptamer and found that sgRNA with the MS2 in stem loop 4 yielded the highest editing efficiency (Fig. 3d, e). However, adding a second MS2 aptamer in stem loop 4 did not further enhance editing efficiency (Fig. 3f). To optimize the linker connecting MCP and TadA8e, we generated the MCP–TadA8e constructs comprising a central XTEN linker flanked by various repeats of the flexible “GSSG” motif (Supplementary Table 3). However, in 3 endogenous sites with TTTR PAMs, we observed no obvious difference in editing efficiencies or in editing windows among these constructs (Supplementary Fig. 10). Thus, we picked our initial constructs bearing a 32 aa linker in the following experiment. Previous studies have demonstrated that introducing mutations into TadA8e can substantially reduce off-target and bystander editing44–46. We therefore introduced mutations into TadA8e of the C1 construct, generating seven variants, as indicated in Fig. 3g. These variants were evaluated at four target sites and the variant dCas12f-P2A-MCP-TadA8e (F148A) showed both high editing efficiency and a narrow editing window mainly restricted to the A3 position (Fig. 3g). This construct was selected for subsequent studies and designated Un1Cas12f1-ABE.

Characterization of evoCas12f-derived base editors and epigenome editor

To generate highly efficient and PAM-expanded miniature base editors, we replaced WT-dUn1Cas12f1 with devoCas12f, creating evoCas12f-ABE. At 11 endogenous sites with non-TTTR PAMs, evoCas12f-ABE demonstrated on average, a 20-fold increase in A-to-G conversion rates (5.96–41.75%) versus Un1Cas12f1-ABE (0.14–5.48%) (Fig. 4a, b). Importantly, evoCas12f-ABE maintained a narrow editing window while achieving broader PAM compatibility (Fig. 4c and Supplementary Fig. 11).

We then compared evoCas12f-ABE with other Cas12f-based ABEs, including dCasMINI-ABE24, eCas12f-ABE33, TaRGET-ABE-C3.130 and miniABE31. Comparison was performed at six endogenous sites with TTTR PAM (Fig. 4d and Supplementary Data 1), which is the optimal PAM for all adenine base editors above. evoCas12f-ABE, eCas12f-ABE and dCasMINI-ABE showed higher A-to-G frequencies than other compact ABEs (Fig. 4d). Although the efficiencies of dCasMINI-ABE and eCas12f-ABE were comparable to evoCas12f-ABE at these targets, evoCas12f-ABE displayed the narrowest editing window. TaRGET-ABE-C3.1, an ABE derived from CWCas12f, showed lower editing efficiency but a broader editing window compare to evoCas12f-ABE, while another Un1Cas12f1 derived miniABE demonstrated minimal activity (Fig. 4d). To further characterize evoCas12f-ABE, we analyzed its A-to-G conversion efficiencies across positions in the above 11 non-TTTR PAM and 6 TTTR PAM targets. The editing window of evoCas12f-ABE is predominantly localized to positions A3 and A4 (Fig. 4e). Overall, evoCas12f-ABE demonstrated superior performance in both efficiency and editing specificity compared to other compact Cas12f-based ABEs. These results suggest that the RNA-protein recruitment strategy confers an additional advantage by reducing bystander editing relative to direct fusion architectures (Fig. 4f). Next, we compared Cas-independent DNA off-target editing of evoCas12f-ABE and other compact Cas12f-based ABEs using the R-loop assay47. All ABEs, except for eCas12f-ABE, induced low (<3%) Cas-independent off-target editing at all six orthogonal SaCas9 sites, with evoCas12f-ABE displaying the highest on-target editing (Fig. 4g).

To generate a miniature CBE, we replaced TadA8e with our previously reported hA3A-D131P, a highly efficient and accurate variant of human APOBEC3A48. We compared C-to-T conversion efficiencies at different positions across two TTTR PAM targets. miniCBE31 produced the highest C-to-T conversion rate (~10%) at position C3, with minimal editing observed at other positions. In contrast, evoCas12f-CBE produced much higher editing efficiency, reaching up to 40% (Fig. 4h). Furthermore, evoCas12f-CBE exhibited superior C-to-T conversion efficiencies at non-canonical PAM targets compared to mini-CBE (Fig. 4i).

The CRISPR-based transcriptional activation system has been shown to be a promising strategy for gene therapy49,50. Encouraged by the success of generating evoCas12f-ABE/CBE, we developed a transcriptional activator via replacing the TadA8e with transcriptional activation domain of P65 and HSF1 (Fig. 4j). We first optimized the relative positions of P65, HSF1, MCP and dUn1Cas12f1 domains, as well as the copy numbers of MS2, assessing activation efficiency using a TRE3G-GFP HEK293T reporter cell line (Supplementary Fig. 12 a–d). The optimal configuration—sgRNA with 5′-MS2+S2-MS2 (Fig. 3d and Supplementary Fig. 12a and b) and construct with TA-N2 architecture—achieved the highest GFP-positive rate (81.53 ± 0.64%) (Supplementary Fig. 12c and d). Subsequently, we replaced the dUn1Cas12f1 component with the devoCas12f, generating an evoCas12f based transcriptional activator—evoCas12fa. We compared the activation activity of four Cas12f transcriptional activation systems (evoCas12fa, SminiCRa32, dCasMINI-VPR24, and AsCas12f-HKRA-MPH43) in the TRE3G-GFP HEK293T reporter cell line. dCasMINI-VPR achieved the highest GFP-positive rate, reaching 88%, followed by evoCas12fa, at 72.8%, slightly higher than that of AsCas12f-HKRA-MPH (71.6%), while the activation efficiency of SminiCRa was the lowest, at only 56.4% (Fig. 4k). To validate evoCas12fa at endogenous loci, we selected 6 target sites across 4 genes: ASCL1, CD2, HBB2, and IFNG. 4 of 6 sites were with TTTR PAMs which were previously used to activate ASCL1 and IFNG24. The other 2 sites were intentionally designed to be preceded by non-canonial PAMs (ATCA and TTCA). Compared with TA-N2 architecture, evoCas12fa demonstrated much higher transcriptional activation activity at the sites with TTTR PAMs, with the highest activation of 4734 fold. Notably, although evoCas12fa showed reduced efficiency at ASCL1-TTCA and HBB-ATCA sites compared to other TTTR PAM targets, the wild type activator (TA-N2) hardly induced gene activation at these loci (Supplementary Fig. 12e). Thus, evoCas12fa outperforms the wild type activation activator (TA-N2) in both efficiency and targeting range.

Precise correction of pathogenic mutations by evoCas12f-ABE and evoCas12f-CBE

To demonstrate the potential of evoCas12f-ABE/CBE, we performed an in-silico survey of ClinVar pathogenic SNVs under editor-specific activity-window and PAM constraints. PAM expansion with evoCas12f increased the ABE targetable fraction from 1.84% to 26.47% (≈14.4-fold) and the CBE targetable fraction from 3.21% to 54.01% (≈16.8-fold) (Fig. 5a). Thus, evoCas12f-ABE/CBE markedly enlarges the pool of variants theoretically correctable relative to WT-Un1Cas12f1 based editors.

To investigate the therapeutic potential of evoCas12f-ABE and evoCas12f-CBE, we designed four sgRNAs with non-TTTR PAMs to correct pathogenic SNVs in ACVRL1 (associated with hereditary hemorrhagic)49, LAMA2 (associated with muscular dystrophy)50, GAA (associated with Pompe disease)51 and NOTCH3 (associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)52. Delivery of evoCas12f-ABE or evoCas12f-CBE with their respective sgRNAs into stable pathogenic SNV cell lines achieved precise correction with efficiencies of 23.1% (ACVRL1), 30.5% (LAMA2), 26.3% (GAA), and 34.7% (NOTCH3) (Fig. 5b). The observed editing efficiencies and specificity, coupled with their flexible PAM requirements, underscore the promise of these tools for gene therapy.

Efficient generation of genetic disease model via evoCas12f

To explore the potential of evoCas12f in vivo, we targeted the tyrosinase (Tyr) gene to generate a mouse model for albinism. We designed 8 sgRNAs in exon 1 of mouse Tyr gene and tested them in NIH/3T3 cells (Fig. 5c and Supplementary Data 1). evoCas12f exhibited dramatically higher cleavage activity than WT-Un1Cas12f1 across all targets with diverse PAMs, particularly at Tyr-sg1, sg3, sg4, and sg6, which were minimally edited by WT-Un1Cas12f1. Among these, Tyr-sg7 demonstrated the highest cleavage efficiency (Fig. 5d) and was selected for embryo microinjection. A mixture of in vitro-transcribed sgRNA and evoCas12f mRNA was microinjected into one-cell-stage embryos of C57BL/6J mice. Of the 26 F0 mice generated, 17 carried modified Tyr alleles, with editing efficiencies ranging from 6.3% to 99.9% (Fig. 5e). Three albino mice displayed near-homozygous phenotypes (Fig. 5f). Genotyping confirmed that 5 of them contained over 90% mutation rates, and 10 of them showed about 50% editing efficiency (Supplementary Table 4). Notably, all 17 mice with modified Tyr alleles exhibited ≤4 types of indels, suggesting that evoCas12f achieves highly efficient cleavage and completes editing by the 2-cell stage. These results highlight the utility of evoCas12f for rapid and precise generation of animal models.

Specificity analysis and optimization of evoCas12f

To evaluate the specificity of evoCas12f, we firstly performed sgRNA mismatch assays using sgRNAs with single or dual-base mismatches across the protospacer region. The mismatch tolerance pattern of evoCas12f closely resembled those of WT-Un1Cas12f1 and CasMINI. evoCas12f activity was notably compromised when single mismatches occurred at the positions 1–5 or 18–20 within the protospacer, or when dual mismatches were present at positions 19–20. However, the activities of all Cas12f variants were fully abolished when a mismatch occurred at positions 6-17 within the protospacer (Fig. 6a).

Next, we conducted GUIDE-seq51 to further evaluate the off-target events of evoCas12f at two endogenous sites (Fig. 6b). We quantified off-target editing sites where reads exceeded 1% of the total. evoCas12f demonstrated higher total read counts than WT-Un1Cas12f1, indicating enhanced enzymatic activity. At the TTTG-12 site, both WT-Un1Cas12f1 and evoCas12f exhibited only background-level off-target editing, confirming evoCas12f’s preserved fidelity. However, at the ATTA-4 site, evoCas12f showed elevated off-target activity, evidenced by a higher percentage of off-target reads. Further sequence analysis revealed that mismatches predominantly occurred at positions 1-5 and 18-20 downstream of the PAM, consistent with our mismatch assay results. Additionally, PAM analysis at off-target sites revealed a T>C preference at the second base, aligning with our endogenous targets. These results underscore the importance of selecting targets with unique sequences or ≥3 mismatches within the seed region to minimize off-target effects in precision applications.

While evoCas12f broadens the target range and improves editing efficiency, its elevated off-target activity remains a concern. Prior studies reported that disrupting non-specific contacts between target DNA and positively charged or polar residues in Cas proteins enhances specificity52,53. Inspired by this and based on the Un1Cas12f1 crystal structure, we generated 26 evoCas12f variants carrying single mutation and tested them at known off-target sites. 6 of the variants showed improved specificity and comparable activity over evoCas12f at the CLIC4 (TTTA-21) target site (Supplementary Fig. 13). To further improve specificity, we introduced two or three of these six mutations into evoCas12f and evaluated the on-target/off-target efficiency. Most of the combined mutants retained on-target activity while substantially reduced off-target effects (Fig. 6c). Compared to evoCas12f, introducing K179A/Y232A mutation in evoCas12f (evoCas12f-2A) reduced off-target editing and increased on-target activity, while additional R258A substitution (evoCas12f-3A) showed similar effects (Fig. 6c), and it was further confirmed through testing of three endogenous target sites (Fig. 6d). These data suggest that introduction of additional mutations could substantially reduce potential off-target effects for more precise editing.

Methods
Ethics statement
Our research complies with all relevant ethical regulations. All animal experiments were approved by the Animal Experiment Ethics Committee of East China Normal University (approval number: m20240305).

Plasmid construction
Human codon-optimized Un1Cas12f1, ge4.1, and P65-HSF1 were synthesized by GENEWIZ. Point mutations in Un1Cas12f1 were introduced via PCR and cloned into the pCMV vector using ClonExpress MultiS One Step Cloning Kit (Vazyme). sgRNA oligos were subjected to a 5-minute denaturation at 95°C, followed by gradual cooling to room temperature to form double strands, which were then ligated into the U6-ge4.1-Ef1α-EGFP vector treated with BbSI restriction enzyme using T4 ligase. All sequences are listed in Supplementary Data 2. The Un1Cas12f1 mutation library oligo was synthesized by GenScript, and the library fragments were amplified using the NEBNext® Q5 Hot Start HiFi PCR kit and purified with the QIAquick gel extraction kit. The purified fragments were assembled with Gibson Assembly Master Mix (NEB) into the PET-28a plasmid backbone treated with restriction enzymes using. PAM-target sequence oligos were annealed using the same method for sgRNA and ligated to the p11-LacY-wtx1 vector treated with XbaI and HindIII restriction enzymes using T4 ligase. Lentiviral vector plasmids for generating stable HEK293T cell lines were constructed using the lentiCRISPRv2 (Addgene #52961), by inserting an ~80 bp gene fragment containing pathogenic SNVs from the ClinVar database and an Ef1a promoter-driven DsRed expression cassette.

sgRNA design
All sgRNAs targeting endogenous sites were listed in Supplementary Data 1 along with their PAM sequences. All endogenous sites with TTTR PAMs and a portion of endogenous sites with non-canonical PAM were previously validated24,25,30,35, while the remaining targets, including targets for Tyr in animal studies were randomly designed. For endogenous gene activation, sgRNAs are designed within the 250 base pairs relative to transcription start site.

Bacterial positive selection assay
E. coli strain BW25141 (λDE3) was purchased from Bioneer. The screening plasmid containing an arabinose inducible ccdb gene was first electroporated into the E. coli and maintained under Ampicillin selection. The Un1Cas12f1 mutant library (including sgRNA) was electroporated into E. coli for screening. Cells were then incubated in pre-warmed 2× YT medium at 37°C for 1 h to allow for full recovery. The entire cell suspension was added to a conical flask containing 100 mL of liquid 2× YT medium (supplemented with 50 μg/mL kanamycin and 0.5 mM IPTG) and incubated at 37°C with shaking at 250 rpm for 6 h. Four milliliters of the culture were spread evenly onto four 25×25 cm square petri dishes (containing 2× YT solid medium, 50 μg/mL kanamycin, and 10 mM arabinose). Plates were incubated overnight at 37°C. The following day, colonies were collected, and plasmids were extracted using a HiPure BAC EF Maxi Kit (Megan). The extracted plasmids served as templates to amplify the library fragments, which were subsequently sequenced via next-generation sequencing (NGS) to identify mutant variants.

Cell culture, cell transfection, and genomic DNA preparation
HEK293T (ATCC CRL-3216) and NIH/3T3 (ATCC CRL-1658) cell lines were purchased from ATCC. The GFP-expressing cell line was a gift from Professor Yongming Wang (Fudan University). All cells were cultured at 37°C in a 5% CO2 atmosphere. Complete growth medium consisted of DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. For transfection, HEK293T cells were seeded at a density of 200,000 cells per well in 24-well plates with 500 μL of complete medium and incubated overnight. When cells reached ~80% confluency, 750 ng of pCMV-Un1Cas12f1 and 250 ng of U6-ge4.1-sgRNA-Ef1a-GFP were co-transfected using PEI the manufacturer’s instructions (Polysciences). Three days post-transfection, GFP-positive cells were sorted using a FACSAria III (BD Biosciences) and collected (Supplementary Fig. 14). Genomic DNA was extract by adding 20 μL of Quick Extract™ DNA Extraction Solution, followed by vortexing for 20 s, incubation at 65°C for 6 min, a second vortexing for 20 s, and final incubation at 98°C for 2 min.

RNA preparation, reverse transcription, and qPCR
GFP-positive cells were sorted using a FACSAria III (BD Biosciences) and collected (Supplementary Fig. 14). Total RNA was extracted following the instructions of the RaPure Total RNA Micro Kit (Megan). cDNA was prepared using the Hifair® II 1st Strand cDNA Synthesis SuperMix, and real-time quantitative PCR was performed using the Hieff® qPCR SYBR Green Master Mix.

Next-generation sequencing and data analysis
NGS primers were designed to ensure PCR products of 140-200 bp in size. The primers incorporated 5′ overhang sequences (forward primer: 5′–ggagtgagtacggtgtgc–3′; reverse primer: 5′–gagttggatgctggatgg–3′). The above PCR products were subjected to a second round of PCR using primers with different barcode sequences. PCR products were mixed and sequenced on Illumina HiSeq platform. All targets information used in this study are detailed in the Supplementary Data 1.

For indel analysis, reads displaying mismatches on both ends of the reference sequence with a frequency under 1% were excluded. Allele quantification was performed by enumerating indel-containing reads which were normalized against the total read count using a custom Cas-analyzer-based script58. For base conversion analysis (A-to-G/C-to-T editing), the frequency of G/T substitutions at targeted A/C bases was calculated as a fraction of total aligned reads, using a custom BE-Analyzer-derived script59.

Lentiviral packaging and stable cell line construction
HEK293T cells were seeded in 6-well plates and transfected at ~80%. A total of 4.5 μg plasmid DNA per well was transfected, comprising 1.5 μg each of the lentiviral vector plasmid, psPAX2, and pMD2.G, using PEI for transfection. After 8 h, the medium was replaced with fresh complete medium. Viral supernatant was collected after 48 h post transfection and filtered through a 0.22 μm filter. For stable cell line establishing, HEK293T cells in 6-well plates were transduced with 100 μL of viral supernatant. After 24 h, the medium was replaced with fresh complete medium. Once the cells were confluent, they were passaged into 10 cm dishes and cultured with complete medium supplemented with 2 μg/mL puromycin. Cell viability was monitored under selection pressure, and the puromycin-containing medium was replaced if excessive cell death occurred a stable cell line was established after two passages.

Calculation of probability mass function for adjacent PAM distances
The calculation method for adjacent PAM distances referred to published articles35,39. PAM sites for each Cas effector on both strands human genome (GRCh38) were scanned and sorted. Distances between adjacent PAMs were computed as: PAM_position_(i+1) – PAM_position_i, in base pairs. Probability Mass Function was constructed as: number of adjacent PAM pairs with distance X / total number of valid adjacent distances of this PAM.

GUIDE-seq assay
GUIDE-seq was carried out according to the established protocols51. Briefly, 1×10⁶ HEK293T cells were electroporated with target plasmids and double-stranded oligodeoxynucleotide (dsODN), followed by 72 h of culture before genomic DNA extraction. The isolated DNA was fragmented, subjected to end-repair and A-tailing, ligated to adapters, and amplified using dsODN-specific primers. Sequencing was performed on an MGI-seq 2000 platform (BGI) in paired-end mode. For data processing, the open-source GUIDE-seq software was employed with proper PAMs. Reads were analyzed using the GUIDE-seq computational pipeline (https://github.com/aryeelab/guideseq), followed by downstream analysis.

Animal studies
6–10-week-old female SPF-grade C57BL/6 strain and ICR strain mice purchased from Laboratory Animal Center in Shanghai were housed at the Experimental Animal Center of East China Normal University. All mice were housed in a specific pathogen-free facility on 12 h light/dark cycles with a temperature between 20°C and 22°C and humidity of 40–60%. All mice had ad libitum access to food and water in standard cages. All animal experiments were approved by the Animal Experiment Ethics Committee of East China Normal University (approval number: m20240305). evoCas12f mRNA was in vitro transcribed using the MEGAscript™ T7 AM1334 kit, and sgRNA was synthesized by GenScript. The sgRNA and mRNA were mixed at a final concentration of 100 ng/ul and centrifuged at 14,800 × g for 60 min at 4°C. The samples were injected into the cytoplasm of one-cell-stage embryos and then transferred into the uterus of surrogate mothers.

Statistical analysis and reproducibility. Data analysis was performed using GraphPad Prism software (version 9.3). Results are expressed as mean ± standard deviation. Statistical evaluations included at least three biologically independent experiments or samples. Comparisons between groups were analyzed using unpaired two-tailed Student’s t-tests, with a P value < 0.05 considered statistically significant. No data were excluded from the analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports the engineering of Un1Cas12f1 to generate evoCas12f with broadened PAM recognition (NTNR/NYTR), improved on-target activity, and adaptation for base editing and transcriptional activation. Given the cargo constraints of in vivo delivery and the typical PAM limitations of Type V Cas12f nucleases, these advances are timely and of broad interest. The study is comprehensive, well executed, and of high relevance to the field. Addressing the points below would further strengthen the work prior to publication.\n1. The authors note that introducing arginine residues is a common strategy to strengthen DNA–protein interactions in Cas protein engineering. Your structural analysis and functional data identify Q244R as a key driver of broadened PAM compatibility via new hydrophilic interactions and salt bridges. By contrast, the other beneficial mutations (N133S, D143S, E151V, L152S) are non-arginine substitutions. Given the demonstrated benefit of Q244R and precedent for arginine substitutions, it would be informative to test arginine at the other identified sites (N133, D143, E151, L152), individually and in combination (for example, N133R, D143R, E151R, L152R). Alternatively, please provide a structural rationale if such substitutions are predicted to clash, destabilize the protein, or increase off-target activity. Even negative results would help refine design principles.\n2. Kim et al. (PMID: 36797406) reported a highly optimized Un1Cas12f variant (CWCas12f). A direct head-to-head comparison of evoCas12f and CWCas12f across targets representing different PAM contexts would clarify the magnitude and breadth of evoCas12f’s PAM flexibility and activity advantages.\n3. Prior work shows that the linker between the nuclease and deaminase domains can markedly affect base editor efficiency and specificity. Although this study uses an MS2–MCP recruitment strategy, varying the linker composition and length between MCP and TadA could further improve performance. Please consider testing a small panel of flexible and semi-rigid linkers of different lengths to identify optimal configurations for both ABE and CBE formats.\n4. While GUIDE-seq and mismatch-tolerance assays are appropriate for profiling DNA cleavage specificity, they do not capture promiscuous deamination on ssDNA, which is a key risk for base editors. To more directly assess evoCas12f-ABE and Un1Cas12f1-ABE specificity, we recommend performing an R-loop assay to compare R-loop formation and stability, which are closely linked to off-target deamination potential. This would provide a more direct assessment than DSB-based methods of how the engineered nuclease affects base editor specificity.\n5. evoCas12f retains a preference for T at the second PAM position. Please discuss, based on your structural model, the molecular basis for this residual bias and whether targeted mutations might further relax it. Identifying candidate residues and providing preliminary modeling or mutational evidence would be valuable.", "reviewer_2": "The study “Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope” by Huo, Mei, Zhang, and colleagues reports the development of evoCas12f, an engineered Cas12f ortholog with expanded PAM recognition. The work addresses an important goal for the genome editing field: creating compact, efficient editors with broad targeting scope. The authors employ a bacteria-based positive selection screen to identify Cas12f variants with broadened PAM compatibility, ultimately engineering evoCas12f with both improved catalytic activity and relaxed PAM requirements. They further apply evoCas12f as a nuclease, base editor, and epigenome editor, and introduce additional mutations aimed at reducing off-target activity while preserving high on-target efficiency.\nWhile the strategy and enzyme engineering are of clear interest, the manuscript requires substantial revision to support accurate interpretation of the results. The relative advantages of evoCas12f compared with existing engineered or wild-type Cas12 enzymes are not evident from the presented data. Moreover, the chosen applications do not fully highlight the stated benefits of Cas12f’s small size and expanded targeting scope. For example, compactness would be most impactful for in vivo delivery using viral vectors, while broader PAM compatibility could unlock editing of otherwise inaccessible genomic variants. As written, these potential advantages are mentioned but not convincingly demonstrated. Ideally, this manuscript would be extensively rewritten to emphasize the motivation behind the experimental choices made and contextualize the results of the experimental choices such that the utility of evoCas12f becomes evident. The following comments should clarify the recurring issues that underlie the manuscript and may guide a more comprehensive revision.\nMajor comments:\n1) The manuscript makes comparisons to other engineered Cas12 proteins but is missing any data making comparisons to arguably the most relevant enzyme for comparison: enAsCas12f (PMID: 37776859). The authors cite this work and are thus aware of it. It seems like a major oversight excluding this enzyme from the comparative benchmarking of evoCas12f.\n2) The applications of the engineered enzyme seem poorly chosen and undermine the utility of evoCas12f. Specifically, the mouse model generation and epigenome editing do not seem to be uniquely enabled by evoCas12f. Knocking out the Tyr gene could be readily accomplished by a wide variety of Cas9 or Cas12 enzymes. Indeed, genome editing of mouse embryos is very well-established. Based on the data presented, it is unclear why any researcher should consider adopting evoCas12f over the other established enzymes. In addition, epigenome editing is not typically constrained by PAM availability—the targeting area that permits gene activation is traditionally quite large. While the authors speculate that the lack of potency of conventional systems is likely due to “rigid target site” requirements, the data presented in Figure 4k are insufficient to support this. To more adequately make a case for transcriptional activation being uniquely enabled by evoCas12f, a more thorough comparison must be made to the alternative enzymes. For example, if all viable guide RNAs targeting the area upstream of the TSS are exhaustively tested for alternative enzymes (including the enCas12f enzyme) is evoCas12f still a top-performer?\n3) In contrast to transcriptional activation and mouse model generation, effective base editing is extremely sensitive to PAM availability and is thus an excellent use case for evoCas12f. The base editing data in the manuscript is well-done and easily interpretable. This section is a clear strength of the manuscript, and should be expanded upon. For example, the authors could identify a mutation that would be difficult to correct with existing Cas12 enzymes but easily targetable with evoCas12f. This could even be applied in silico to pathogenic mutations listed on ClinVar, enumerating what would likely be targetable with evoCas12f but not wildtype Un1Cas12f1, though an actual application in a primary cell line or animal model would be more convincing. Effectively, the varied applications of evoCas12f in this manuscript are either poorly motivated and unlikely to be of interest or adopted by the broader genome editing community or are well-motivated but require more thorough evaluation.\n4) There is insufficient methodological detail to effectively evaluate experimental design and conclusions. A subsection explaining how guide RNAs were selected is imperative. For example, was every viable guide RNA used in the transcriptional activation experiments? Were guides designed within a certain window relative to the TSS? How were the 7 guides chosen targeting exon 1 of the mouse Tyr gene? Most experiments described are very difficult to accurately evaluate given the lack of methodological detail in the results or methods subsections.\n5) The results subsections consistently lack sufficient detail to interpret results. For example, something called “TaRGRT-C3.1” is mentioned as having a broader editing window compared to evoCas12f-ABE. What is TaRGRT-C3.1? The authors state “miniABE demonstrated minimal activity”, again with no explanation as to what “miniABE” is. This is a consistent issue with the manuscript. Even when clarity is provided regarding what is being tested, there is no contextual information to help a reader interpret the results. ABE experiments comparing the activity of evoCas12f to other enzymes are performed at 6 sites with TTTR PAMs. What are the PAMs of the other enzymes that were tested? Should I expect these alternative enzymes to have any activity on a target-site with a TTTR PAM? There are no detailed descriptions of what these enzymes are or citations to the relevant literature that describes them. Without these details, it is hard to appreciate or ascertain any relative benefit of evoCas12f.\nMinor comments:\n1) Inconsistent naming is used throughout the manuscript and figures for the adenine base editors. Sometimes the specific deaminase used (TadA8e) is specified, and sometimes it’s just referred to as “ABE”. This should be made consistent to avoid confusion.\n2) It is unclear what PHM stands for when referring to the transcriptional activator versions of evoCas12f.\n3) The epigenome editing targets (HBB-ATCA and ASCL1-TTCA) could be more clearly defined upon first mention. I assume HBB and ASCL1 are referring to genes while the four following letters are referring to the PAM sequence. This should be made clearer, alongside some justification for why these were chosen (in line with comments above regarding methodological unclarity).\n4) Mentioning fold increases (like “evoCas12f achieved a 99.7-fold increase”) is unnecessary and potentially misleading when the starting activity is near 0%. Reporting the absolute editing efficiencies measured is sufficient for a reader to appreciate and interpret the results.\n5) Panel F in Figure 5 seems unnecessary or could be moved to a Supplementary Table. The relevance of the exact editing outcomes is unclear. These are specific to this edit (which is a test edit) and the general conclusion that you can effectively generate indels is well represented throughout the other data in this figure and the rest of the manuscript."}, "author_response": "Response to the reviewers:\nReviewer #1 (Remarks to the Author):\nThis manuscript reports the engineering of Un1Cas12f1 to generate evoCas12f with\nbroadened PAM recognition (NTNR/NYTR), improved on-target activity, and adaptation for\nbase editing and transcriptional activation. Given the cargo constraints of in vivo delivery\nand the typical PAM limitations of Type V Cas12f nucleases, these advances are timely\nand of broad interest. The study is comprehensive, well executed, and of high relevance\nto the field. Addressing the points below would further strengthen the work prior to\npublication.\nMajor comments:\nComment 1. The authors note that introducing arginine residues is a common strategy to\nstrengthen DNA–protein interactions in Cas protein engineering. Your structural analysis\nand functional data identify Q244R as a key driver of broadened PAM compatibility via new\nhydrophilic interactions and salt bridges. By contrast, the other beneficial mutations\n(N133S, D143S, E151V, L152S) are non-arginine substitutions. Given the demonstrated\nbenefit of Q244R and precedent for arginine substitutions, it would be informative to test\narginine at the other identified sites (N133, D143, E151, L152), individually and in\ncombination (for example, N133R, D143R, E151R, L152R). Alternatively, please provide\na structural rationale if such substitutions are predicted to clash, destabilize the protein, or\nincrease off-target activity. Even negative results would help refine design principles.\nResponse: We thank for the reviewer’s suggestion. In the revised version, we’ve mutated\neach of the N133, D143, E151, L152 to arginine individually as well as in combination. We\ndiscovered that none of the individual mutation had obvious superior editing efficiency.\nWhen we combine the 5-arginine together, the resulting construct showed lower editing\nefficiencies at endogenous PAM sites of NTTR and TNTR (Supplementary Fig. 2).\nTherefore, although positively charged argine might increase the affinity of the protein to\nDNA backbone, it is not always be the best strategy for higher editing activity, due to\npotential structural constrains. We’ve added a figure (Supplementary Fig. 2) to the result\nand made corresponding modification in the paper. Please see line: 145-152 and below:\n“Arginine (Arg) substitutions are often used to strengthen DNA–protein interactions. Motivated by\nthe benefit of Q244R, we introduced individual Arg substitutions at N133, D143, L152, and E151\nand also evaluated combinatorial variants. However, the results indicated that D143S, L152S,\nN133S, and E151V each had generally higher editing efficiency than their to-arginine counterparts\nat the corresponding PAM endogenous sites. (Supplementary Fig. 2 a-d) Moreover, the five-Arg\nconstruct (N133R/D143R/E151R/L152R/Q244R) exhibited consistently lower activity on NTTR\nand TNTR PAM sites (Supplementary Fig. 2e)”\nSupplementary Fig. 2 | Activity comparison of the screened mutations and their arginine\nsubstitutes. a-e, The indel frequency of the obtained mutations and arginine substitutes at the\ncorresponding PAM endogenous sites. Values and error bars represent mean ± SD from three\nindependent experiments.\nComment 2.Kim et al. (PMID: 36797406) reported a highly optimized Un1Cas12f variant\n(CWCas12f). A direct head-to-head comparison of evoCas12f and CWCas12f across\ntargets representing different PAM contexts would clarify the magnitude and breadth of\nevoCas12f’s PAM flexibility and activity advantages.\nResponse: In response to the reviewer’s question, we performed head-to-head\ncomparisons between evoCas12f and two CWCas12f variants (S188Q+Q272K and\nS188K+Q272K) across 15 targets representing NTNR and TVTR PAMs. In our results,\nevoCas12f showed higher editing efficiencies at 12/15 of the tested sites. Notably,\nCWCas12f exhibited minimal activities on TATR PAMs, while evoCas12f had efficiencies\nupto 35%. These results support broader PAM activity and higher overall efficiency of\nevoCas12f. We added these results in Supplementary Fig. 8b and made corresponding\nmodifications in the article. Please see line: 212-216 and below.\n“We further evaluated evoCas12f against other miniature Cas12 members including\nCasMINI24,CWCas12f30 variants, AsCas12f variants43, enOsCas12f127 and enRhCas12f127. As\nshown in Supplementary Fig. 8, Across 47 endogenous targets spanning five compact Cas12f\nfamilies, evoCas12f displayed generally superior or comparable activity.”\nSupplementary Fig. 8b | Comparison of indel frequencies between evoCas12f and other\nengineered Cas12f editors. b, CWCas12f (S188Q+Q272K), CWCas12f (S188K+Q272K), and\nevoCas12f.\nComment 3. Prior work shows that the linker between the nuclease and deaminase\ndomains can markedly affect base editor efficiency and specificity. Although this study uses\nan MS2–MCP recruitment strategy, varying the linker composition and length between\nMCP and TadA could further improve performance. Please consider testing a small panel\nof flexible and semi-rigid linkers of different lengths to identify optimal configurations for\nboth ABE and CBE formats.\nResponse: We appreciate the suggestion and systematically evaluated linker length in our\nMS2–MCP recruitment design. Specifically, we generated MCP–TadA8e constructs\ncomprising a central XTEN linker flanked by repeats of the flexible “GSSG” motif\n(Supplementary Table 4). However, in the 3 endogenous sites with TTTR PAMs, we\nobserved no obvious difference in editing efficiencies or in editing windows among these\nconstructs (Supplementary Fig 10). These results indicate that MCP–TadA8e has sufficient\nconformational flexibility to engage substrates without further gains from length extension.\nWe therefore proceeded with the original 32aa linker for ABE experiments. We’ve add the\nexperiment data in Supplementary Fig 10 and made corresponding modifications. Please\nsee line: 240-246 and below.\n“To optimize the linker connecting MCP and TadA8e, we generated the MCP–TadA8e constructs\ncomprising a central XTEN linker flanked by various repeats of the flexible “GSSG” motif\n(Supplementary Table 4). However, in 3 endogenous sites with TTTR PAMs, we observed no\nobvious difference in editing efficiencies or in editing windows among these constructs\n(Supplementary Fig. 10). Thus we picked our initial constructs bearing a 32 aa linker in the\nfollowing experiment.”\nSupplementary Fig. 10 | Determination of the optimized length of a linker used to connect\ndUn1Cas12f1 and Tad8e.Values and error bars represent mean ± SD from three independent\nexperiments.\nComment 4. While GUIDE-seq and mismatch-tolerance assays are appropriate for\nprofiling DNA cleavage specificity, they do not capture promiscuous deamination on\nssDNA, which is a key risk for base editors. To more directly assess evoCas12f-ABE and\nUn1Cas12f1-ABE specificity, we recommend performing an R-loop assay to compare R-\nloop formation and stability, which are closely linked to off-target deamination potential.\nThis would provide a more direct assessment than DSB-based methods of how the\nengineered nuclease affects base editor specificity.\nResponse: We appreciate the reviewer’s suggestion and performed R-loop assay to\ncompare the off-target activities among evoCas12f-ABE, dCasMINI-ABE, TaRGET-ABE-\nC3.1, miniABE and eCas12f-ABE. As a result, all ABE-base editors, except for eCas12f-\nABE, induced low Cas-independent off-target editing at all six orthogonal SaCas9 sites\n(<3%), while evoCas12f-ABE achieved highest on-target editing activity. Therefore the\nmutations on WT-Un1Cas12f1 does not obviously increase the off-target deamination\npotential. We added this part into the main text in line 278-282 and Fig. 4g. See below.\n“Next we compared Cas-independent DNA off-target editing of evoCas12f-ABE and other compact\nCas12f-based ABEs using the R-loop assay. All ABEs, except for eCas12f-ABE, induced low (<3%)\nCas-independent off-target editing at all six orthogonal SaCas9 sites, with evoCas12f-ABE\ndisplaying the highest on-target editing (Fig. 4g). ”\nFig 4g. | Generating evoCas12f-derived tools based on the MS2-MCP strategy. g, Average on-\ntarget editing versus average off-target editing for Cas12f related ABEs. The y-axis reflects the\naverage on-target A-to-G editing, the x-axis reflects the mean cumulative off-target A-to-G editing\nin orthogonal R-loops assay, editing efficiencies shown represent the mean of three biologically\nindependent replicates.\nComment 5. evoCas12f retains a preference for T at the second PAM position. Please\ndiscuss, based on your structural model, the molecular basis for this residual bias and\nwhether targeted mutations might further relax it. Identifying candidate residues and\nproviding preliminary modeling or mutational evidence would be valuable.\nResponse: We appreciate the reviewer’s suggestion. Guided by the crystal structure of\nCas12f-DNA complex, we identified Y146 and Q197 as residues contacting the second\nPAM base (Supplementary Fig. 6a). We then performed single-site saturation mutagenesis\nat each position and tested editing at six VVTR sites. Substitutions at Y146 uniformly\nabolished activity, indicating a strong Un1Cas12f1 structural constraint at this position. By\ncontrast, Q197K increased activity at 4/6 VTTR sites (Supplementary Fig. 6b). We further\nprofiled Q197K across 40 NNTR endogenous targets and discovered that it boosted\nactivity on NCTR, had minimal effect on NRTR, and reduced activity on NTTR to near WT\nUn1Cas12f1 levels (Supplementary Fig. 6c). Thus, Q197K does not broaden the overall\nNNTR range; rather, it shifts the preference of the second T in PAM to C, yielding a\nspecialized variant, evoCas12f(Q197K), optimized for NCTR targets. We have added a\nfigure (Supplementary Fig. 6) and included the descrption of this experiment in the main\ntext of the article (line: 182-192 ) and see below.\n“To address evoCas12f’s mild preference for T at PAM position 2, structural analysis identified\nY146 and Q197 as contacting the second T of the PAM (Supplementary Fig. 6a). We conducted\nsingle amino acid saturation mutagenesis at each residue and tested editing at 6 VVTR sites. The\nresults showed that any substitution at Y146 abolished its activity, whereas Q197K increased its\nactivity at 4/6 VTTR PAM sites (Supplementary Fig. 6b). We then evaluated Q197K at 40 NNTR\nendogenous loci and found that Q197K boosts activity on NCTR PAMs, has minimal effect on\nNRTR, and reduces activity on NTTR to near WT Un1Cas12f1 levels (Supplementary Fig. 6c).\nCollectively, Q197K does not expand evoCas12f’s overall NNTR targeting range but shifts the\npreference of the 2nd position from T to C. Thus evoCas12f (Q197K) is best considered a specialized\nvariant for NCTR targets.”\nSupplementary Fig. 6 | Further optimization evoCas12f based on the crystal structure. a,\nSchematic diagram of the partial crystal structure of Un1Cas12f1. Light blue represents the\nUn1Cas12f1 protein structure (PDB:7C7L), light purple represents the DNA target strand, orange\nrepresents the non-target strand, the T·A base pair at the second position of the PAM is highlighted\nin red, and the residues interacting with it are highlighted in dark blue.b, Heatmaps illustrating indel\nfrequency ratio of each mutant to evoCas12f at corresponding genomic loci. The values represent\nthe average of 3 independent experiments. c, Comparative analysis of PAM nucleotide selectivity\nat position 2 between Un1Cas12f1, evoCas12f and evoCas12f+Q197K, editing efficiencies of\nUn1Cas12f1 and evoCas12f are from Fig. 2a. Each dot represents the mean of three independent\nexperiments at the same genomic locus, n.s. not significant, * p < 0.05.\nReviewer #2 (Remarks to the Author):\nThe study “Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity\nand targeting scope” by Huo, Mei, Zhang, and colleagues reports the development of\nevoCas12f, an engineered Cas12f ortholog with expanded PAM recognition. The work\naddresses an important goal for the genome editing field: creating compact, efficient\neditors with broad targeting scope. The authors employ a bacteria-based positive selection\nscreen to identify Cas12f variants with broadened PAM compatibility, ultimately\nengineering evoCas12f with both improved catalytic activity and relaxed PAM\nrequirements. They further apply evoCas12f as a nuclease, base editor, and epigenome\neditor, and introduce additional mutations aimed at reducing off-target activity while\npreserving high on-target efficiency.\nWhile the strategy and enzyme engineering are of clear interest, the manuscript requires\nsubstantial revision to support accurate interpretation of the results. The relative\nadvantages of evoCas12f compared with existing engineered or wild-type Cas12 enzymes\nare not evident from the presented data. Moreover, the chosen applications do not fully\nhighlight the stated benefits of Cas12f’s small size and expanded targeting scope. For\nexample, compactness would be most impactful for in vivo delivery using viral vectors,\nwhile broader PAM compatibility could unlock editing of otherwise inaccessible genomic\nvariants. As written, these potential advantages are mentioned but not convincingly\ndemonstrated. Ideally, this manuscript would be extensively rewritten to emphasize the\nmotivation behind the experimental choices made and contextualize the results of the\nexperimental choices such that the utility of evoCas12f becomes evident. The following\ncomments should clarify the recurring issues that underlie the manuscript and may guide\na more comprehensive revision.\nMajor comments:\nComment 1. The manuscript makes comparisons to other engineered Cas12 proteins but\nis missing any data making comparisons to arguably the most relevant enzyme for\ncomparison: enAsCas12f (PMID: 37776859). The authors cite this work and are thus\naware of it. It seems like a major oversight excluding this enzyme from the comparative\nbenchmarking of evoCas12f.\nResponse：We thank the reviewer for highlighting this point. We agree that enAsCas12f\n(PMID: 37776859) represents a particularly relevant benchmark. Accordingly, we have\nincorporated direct comparisons between evoCas12f and two enAsCas12f variants (YHAM\nand HKRA) on the same set of matched target sites (Supplementary Fig. 8c). The result\nshowed that AsCas12f-HKRA displayed comparable to or slightly higher indel frequencies\nthan evoCas12f at T-rich PAMs, while evoCas12f outperformed AsCas12f-YHAM at 11 of\n12 targets. We have revised the main text . Please see lines 212–216 and below.\nSupplementary Fig. 8c | Comparison of indel frequencies between evoCas12f and other\nengineered Cas12f editors. c, AsCas12f-YHAM, AsCas12f-HKRA, and evoCas12f. Values and\nerror bars represent mean ± SD from three independent experiments.\nComment 2. The applications of the engineered enzyme seem poorly chosen and\nundermine the utility of evoCas12f. Specifically, the mouse model generation and\nepigenome editing do not seem to be uniquely enabled by evoCas12f. Knocking out the\nTyr gene could be readily accomplished by a wide variety of Cas9 or Cas12 enzymes.\nIndeed, genome editing of mouse embryos is very well-established. Based on the data\npresented, it is unclear why any researcher should consider adopting evoCas12f over the\nother established enzymes.\nIn addition, epigenome editing is not typically constrained by PAM availability—the\ntargeting area that permits gene is traditionally quite large. While the authors speculate\nthat the lack of potency of conventional systems is likely due to “rigid target site”\nrequirements, the data presented in Figure 4k are insufficient to support this. To more\nadequately make a case for transcriptional activation being uniquely enabled by evoCas12f,\na more thorough comparison must be made to the alternative enzymes. For example, if all\nviable guide RNAs targeting the area upstream of the TSS are exhaustively tested for\nalternative enzymes (including the enCas12f enzyme) is evoCas12f still a top-performer?\nResponse：We appreciate the reviewer’s thoughtful critique and agree that we should\nemphasize practical advantages rather than exclusive enablement. For the evoCas12f-\nderived transactivator, our aim is not to claim that evoCas12f is the only nuclease capable\nof transcriptional activation. Rather, we highlight that its relaxed PAM profile increases the\ndensity of placeable guides within promoter-proximal, position-dependent windows,\nthereby raising the likelihood of identifying potent sites and enabling multiplexed gRNA–\nmediated activation, which was reported previously (Nissim et al., PMID: 24837679;\nCampa et al., PMID:31406383; Shaw et al., PMID: 36008396). We apologize for our\nimprecise wording. Our use of the phrase “rigid target site” may have implied an meaning\nwe did not intend to convey and caused unnecessary confusion. We have rewritten this\nsection in the manuscript to clarify the above rationale in both the Results (lines 301–317)\nand Discussion (lines: 447–449). We also added an reporter activation assay so that each\nactivator are compared with same targeted position relative to the TSS (Fig. 4k). The\noriginal Fig. 4k were moved to Supplementary Fig. 12e. See below.\n“an evoCas12f based transcriptional activator—evoCas12fa. We compared the activation activity of\nfour Cas12f transcriptional activation systems (evoCas12fa, SminiCRa, dCasMINI-VPR, and\nAsCas12f-HKRA-MPH) in the TRE3G-GFP HEK293T reporter cell line. dCasMINI-VPR\nachieved the highest GFP-positive rate, reaching 88%, followed by evoCas12fa, at 72.8%,\nslightly higher than that of AsCas12f-HKRA-MPH (71.6%), while the activation efficiency of SminiCRa\nwas the lowest, at only 56.4% (Fig. 4k). To validate evoCas12fa at endogenous loci, we selected 6\ntarget sites across 4 genes: ASCL1, CD2, HBB2, and IFNG. 4 of 6 sites were with TTTR PAMs\nwhich were previously used to activate ASCL1 and IFNG. The other 2 sites were intentionally\ndesigned to be preceded by non-canonial PAMs (ATCA, and TTCA). Compared with TA-N2\narchitecture, evoCas12fa demonstrated much higher transactivational activity at the sites with TTTR\nPAMs, with the highest activation of 4734 fold. Notably, although evoCas12fa showed reduced\nefficiency at ASCL1-TTCA and HBB-ATCA sites compared to other TTTR PAM targets, the wild\ntype activator (TA-N2) hardly induced gene activation at these loci (Supplementary Fig. 12e). Thus,\nevoCas12fa outperforms the wild type activation activator (TA-N2) in both efficiency and targeting\nrange.”\nFig 4k. | Generating evoCas12f-derived tools based on the MS2-MCP strategy. k, GFP\nactivation efficiencies of evoCas12fa, SminiCRa, dCasMINI-VPR and AsCas12f-HKRA-MPH (n\n= 3, mean ± SD).\nIn discussion line 447–449:\n“We believe that the evoCas12f platform, with its high targeting density (1 site per 2 bp), will help to\nraise the likelihood of identifying potent sites and enabling multiplexed gRNA–mediated activation”\nComment 3. In contrast to transcriptional activation and mouse model generation,\neffective base editing is extremely sensitive to PAM availability and is thus an excellent\nuse case for evoCas12f. The base editing data in the manuscript is well-done and easily\ninterpretable. This section is a clear strength of the manuscript, and should be expanded\nupon. For example, the authors could identify a mutation that would be difficult to correct\nwith existing Cas12 enzymes but easily targetable with evoCas12f.\nThis could even be applied in silico to pathogenic mutations listed on ClinVar, enumerating\nwhat would likely be targetable with evoCas12f but not wildtype Un1Cas12f1, though an\nactual application in a primary cell line or animal model would be more convincing.\nEffectively, the varied applications of evoCas12f in this manuscript are either poorly\nmotivated and unlikely to be of interest or adopted by the broader genome editing\ncommunity or are well-motivated but require more thorough evaluation.\nResponse: We thank the reviewer for highlighting base editing as a particularly compelling\nuse case for evoCas12f. We agree and have expanded the base-editing section\naccordingly. We added a genome-wide assessment of ClinVar-listed pathogenic SNVs,\nenumerating variants that are potentially correctable by evoCas12f-ABE/CBE and WT-\nUn1Cas12f-ABE/CBE, given their respective editing window and PAM requirements. As a\nresult, 26.74% of pathogenic SNVs are potentially correctable by evoCas12f-ABE and\n54.01% by evoCas12f-CBE, which was 1.84% and 3.21% for the wild type counterparts\n(Fig 5a). Thus, we believe evoCas12f-ABE/CBE markedly enlarges the pool of variants\ntheoretically correctable relative to WT-Un1Cas12f1 based editors.\nTo further validate this point, we created a series cell-lines bearing pathogenic mutations\nfor hereditary diseases. These mutations are in spacers proceeded by non-canonical PAM\nsequences such TTCA (for NOTCH3 c.1363T>C), TCTA (for GAA c.896T>C), which are\ncorrectable by evoCas12f-ABE or evoCas12f-CBE but inaccessible for WT-Cas12f. We\nexpanded the base editing section in line 318-338 and include the extra data in Fig. 5a and\nb. See below.\n“Precise correction of pathogenic mutations by evoCas12f-ABE and evoCas12f-CBE\nTo demonstrate the potential of evoCas12f-ABE/CBE, we performed an in-silico survey of ClinVar\npathogenic SNVs under editor-specific activity-window and PAM constraints. PAM expansion with\nevoCas12f increased the ABE targetable fraction from 1.84% to 26.47% (≈14.4-fold) and the CBE\ntargetable fraction from 3.21% to 54.01% (≈16.8-fold) (Fig. 5a). Thus, evoCas12f-ABE/CBE\nmarkedly enlarges the pool of variants theoretically correctable relative to WT-Un1Cas12f1 based\neditors.\nTo investigate the therapeutic potential of evoCas12f-ABE and evoCas12f-CBE, We designed\nfour sgRNAs with non-TTTR PAMs to correct pathogenic SNVs in ACVRL1 (associated with\nhereditary hemorrhagic), LAMA2 (associated with muscular dystrophy), GAA (associated with\nPompe disease)51 and NOTCH3 (associated with cerebral autosomal dominant arteriopathy with\nsubcortical infarcts and leukoencephalopathy). Delivery of evoCas12f-ABE or evoCas12f-CBE\nwith their respective sgRNAs into stable pathogenic SNV cell lines achieved precise correction with\nefficiencies of 23.1% (ACVRL1), 30.5% (LAMA2), 26.3% (GAA), and 34.7% (NOTCH3) (Fig. 5b).\nThe observed editing efficiencies and specificity, coupled with their flexible PAM requirements,\nunderscore the promise of these tools for gene therapy.”\nFig. 5 a-b | Applications of evoCas12f and derived base editors. a, Frequencies of genetic\nmutations in ClinVar that could, in principle, be corrected by evoCas12f-ABE and evoCas12f-CBE.\nevoCas12f-ABE targeting A -A ,evoCas12f-CBE targeting C -C and C -C . b, Design of\n3 4 3 4 16 18\nsgRNAs targeting pathogenic SNVs and their correction by base editing. sgRNAs were designed to\ntarget the pathogenic SNVs ACVRL1 c.1436G>A, LAMA2 c.7071G>A , GAA c.896T>C and\nNOTCH3 c.1363T>C. These pathogenic SNVs were rescued using either evoCas12f-ABE or\nevoCas12f-CBE. The histogram shows the correction efficiencies, with the targeted pathogenic\nSNVs highlighted in red. Values and error bars represent the mean ± SD from three independent\nexperiments.\nComment 4.There is insufficient methodological detail to effectively evaluate experimental\ndesign and conclusions. A subsection explaining how guide RNAs were selected is\nimperative. For example, was every viable guide RNA used in the transcriptional activation\nexperiments? Were guides designed within a certain window relative to the TSS? How\nwere the 7 guides chosen targeting exon 1 of the mouse Tyr gene? Most experiments\ndescribed are very difficult to accurately evaluate given the lack of methodological detail in\nthe results or methods subsections.\nResponse: We thank the reviewer for suggesting the need for greater transparency in\ngRNA selection. In the revised manuscript, we have elaborated on the relevant content\nregarding sgRNA design in the sgRNA design subsection of Methods (lines: 485-491), and\nhave listed all the target information in Supplementary table 1 as well as targets for R-loop\nassays and quantitative PCRs in Supplementary tables 2 and 3 respectively. In the animal\nstudies, the seven sgRNAs were randomly designed across the exon 1 of mouse Tyr gene\nin order to identify an optimal guide. See below.\n“sgRNA design\nAll sgRNAs targeting endogenous sites were listed in supplementary table 1 along with their PAM\nsequences. All endogenous sites with TTTR PAMs and a portion of endogenous sites with non-\ncanonical PAM were previously validated24,25,30,35, while the remaining targets including targets for\nTry in animal studies were randomly designed . For enodogenous gene activation, sgRNAs are\ndesigned within the 250 base pairs relative to transcription start site.”\nComment 5. The results subsections consistently lack sufficient detail to interpret results.\nFor example, something called “TaRGRT-C3.1” is mentioned as having a broader editing\nwindow compared to evoCas12f-ABE. What is TaRGRT-C3.1? The authors state\n“miniABE demonstrated minimal activity”, again with no explanation as to what “miniABE”\nis. This is a consistent issue with the manuscript.\nEven when clarity is provided regarding what is being tested, there is no contextual\ninformation to help a reader interpret the results. ABE experiments comparing the activity\nof evoCas12f to other enzymes are performed at 6 sites with TTTR PAMs. What are the\nPAMs of the other enzymes that were tested? Should I expect these alternative enzymes\nto have any activity on a target-site with a TTTR PAM? There are no detailed descriptions\nof what these enzymes are or citations to the relevant literature that describes them.\nWithout these details, it is hard to appreciate or ascertain any relative benefit of evoCas12f.\nResponse: We appreciate the reviewer’s comments and apologize for the typo and the\nlack of necessary information in the text. There’s a typo in “TaRGRT” which should be\n“TaRGET”. The complete name for this adenine base editor is TaRGET-ABE-C3.1, which\nis derived from the reported CWCas12f. miniABE is another adenine base editor derived\nfrom Un1Cas12f1. In the revision we have made corresponding amendment and add\ncitation to these names. In the part of adenine base editor comparison, the TTTR is an\noptimal PAM for all the base editors in the group. Therefore we expect all of them to\nachieve their respective optimal enzymatic activities. We’ve made according amendment\nin the main text of the article line (261-264 and 268-271). See below.\nline 261-264: “We then compared evoCas12f-ABE with other Cas12f-based ABEs, including\ndCasMINI-ABE24, eCas12f-ABE33, TaRGET-ABE-C3.130 and miniABE31. Comparison was\nperformed at six endogenous sites with TTTR PAM (Fig. 4d and Supplementary Table 1), which is\nthe optimal PAM for all adenine base editors above.”\nline 268-271: “TaRGET-ABE-C3.1, an ABE derived from CWCas12f, showed lower editing\nefficiency but a broader editing window compare to evoCas12f-ABE, while another Un1Cas12f1\nderived miniABE demonstrated minimal activity”\nMinor comments:\nComment 1. Inconsistent naming is used throughout the manuscript and figures for the\nadenine base editors. Sometimes the specific deaminase used (TadA8e) is specified, and\nsometimes it’s just referred to as “ABE”. This should be made consistent to avoid confusion.\nResponse: Thank you for the comments. We have revised the manuscript and unified the\nnaming. TadA8e now is just used for describing the deaminase domain in the architecture\ndesign or crystal structural description. ABE is now stands for adenine base editor.\nComment 2. It is unclear what PHM stands for when referring to the transcriptional\nactivator versions of evoCas12f.\nResponse: Thank you for the suggestion. We’ve changed name of the transcriptional\nactivator versions of evoCas12f to evoCas12fa (evoCas12f activator).\nComment 3. The epigenome editing targets (HBB-ATCA and ASCL1-TTCA) could be\nmore clearly defined upon first mention. I assume HBB and ASCL1 are referring to genes\nwhile the four following letters are referring to the PAM sequence. This should be made\nclearer, alongside some justification for why these were chosen (in line with comments\nabove regarding methodological unclarity).\nResponse：Thank you for the comments. In the revised paper we claimed HBB, ASCL1,\nCD2, and IFNG are genes when these names first appeared (lines: 307-310). Also we\nadded gRNA information in thes supplementary table 1.\nComment 4.Mentioning fold increases (like “evoCas12f achieved a 99.7-fold increase”)\nis unnecessary and potentially misleading when the starting activity is near 0%. Reporting\nthe absolute editing efficiencies measured is sufficient for a reader to appreciate and\ninterpret the results.\nResponse: Thank you for the suggestion. In the revised paper we put absolute values in\nthis sentence to avoid misinterpretation. line :170-171.\nComment 5. Panel F in Figure 5 seems unnecessary or could be moved to a\nSupplementary Table. The relevance of the exact editing outcomes is unclear. These are\nspecific to this edit (which is a test edit) and the general conclusion that you can effectively\ngenerate indels is well represented throughout the other data in this figure and the rest of\nthe manuscript.\nResponse: Thank you for the suggestion. In the revised paper we moved these results to\nSupplementary Table 5."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My previous concerns have been fully addressed.", "reviewer_2": "All my concerns have been addressed in the revised manuscript."}, "author_response": ""}]

Decision letter