Improvement of the LbCas12a-crRNA system for e cient gene targeting in plants

Tien Van Vu (  tienvu.agi@gmail.com ) Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University https://orcid.org/0000-0002-6369-7664 Duong Thi Hai Doan Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University Mil Thi Tran Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University Yeon Woo Sung Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University Young Jong Song Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University Jae-Yean Kim (  kimjy@gnu.ac.kr ) Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University https://orcid.org/0000-0002-1180-6232


Introduction
Plant gene targeting (GT) was rst reported in 1988 by Paszkowski and coworkers, although that study only obtained low e ciency of targeting (Paszkowski et al., 1988), and GT usually requires at least one associated selection marker for the practical achievement of GT events (Puchta and Hohn, 2005; Van Vu et al., 2019). Without any targeted DSB and with an antibiotic selection marker, the GT e ciency remained extremely low (Terada et al., 2002). An important improvement was made to plant GT by introducing DSBs at the targeted sites using a pre-inserted I-SceI recognition sequence; this approach ultimately enhanced the GT e ciency up to hundreds of fold (Puchta et al., 1993;Puchta et al., 1996), but the e ciency was still low (i.e., up to 1.83%) even it required two selection markers. The subsequent development of the rst, second and, especially, third generations of site-directed nucleases (SDNs) has revolutionized precision gene-editing technology with the easy customization of targeted DSBs at any site of interest for exchanging donor DNA templates (Shan et al., 2013;Wright et al., 2005;Zhang et al., 2013).
Until recently, SDN-based GT e ciency has been signi cantly enhanced through combinations of SDN complexes and geminiviral replicons, which are autonomously replicative vectors for supplying high Our previous work showed the signi cant improvement of plant GT using geminiviral replicons and LbCas12a, rather than SpCas9 (Vu et al., 2020). More importantly, the activity of LbCas12a nucleases was more temperature-dependent than that of SpCas9 (Malzahn et al., 2019;Vu et al., 2020). Recently, a temperature-tolerant LbCas12a mutant (D156R), known as ttLbCas12a, which exhibited signi cantly improved cleavage activity and hence gene targeting e ciency, was reported in Arabidopsis using oral dipping-based gamete stage transformation (Merker et al., 2020a;Schindele and Puchta, 2020). However, further characterization of ttLbCas12a for somatic cell-based GT systems such as tomato GT, especially in combination with DNA replicons, has not been undertaken. We hypothesized that ttLbCas12a could be utilized to further improve our replicon-based plant GT system through appropriate customization of its nuclease-crRNA complexes. Chemical treatment for suppressing cNHEJ or activating HDR pathways was also tested and validated for practical GT applications in tomato. In this report, we describe a further comparison of LbCas12a and SpCas9 in inducing GT at the same speci c SlANT1 locus. Subsequently, extensive characterization of ttLbCas12a in comparison with LbCas12a is shown at the SlANT1, SlHKT1;2, and SlEPSPS1 loci with or without allele-associated selection markers. Our work demonstrates the signi cance of utilizing the appropriate design of CRISPR/Cas-crRNA complexes, chemical treatments, and favorable experimental conditions to further enhance plant HDR for e cient GT in tomato.

NU7441 treatment enhances LbCas12a-based GT e ciency
A plant genomic DNA DSB may be repaired by two major competing mechanisms, namely, cNHEJ and HDR. The GT approach is based on the HDR mechanism; therefore, to increase its e cacy, blocking the cNHEJ pathway is a good option ( Van Vu et al., 2019). A number of studies have been published regarding the uses of chemical treatments for blocking cNHEJ to enhance GT e ciency in mammals (Chu et al., 2015;Maruyama et al., 2015;Robert et al., 2015;Zhang et al., 2017). However, only limited information regarding the applications of the chemicals to plant GT is currently available. Therefore, we selected SCR7 (an inhibitor of DNA ligase IV), NU7441 (a DNA-dependent protein kinase (DNA-PKcs) inhibitor), and KU0060648 (a dual inhibitor of DNA-PKcs and phosphatidylinositol-3 kinase (PI-3K)) to determine their effects on plant GT. Previously, a replicon-based CRISPR/Cas-mediated targeted DNA insertion system was successfully developed with SlANT1 as a visible marker (Cermak et al., 2015;Vu et al., 2020). We used SpCas9 (pTC217) and LbCas12a (pHR01 and pMR01) carrying GT vectors to assess the effects of chemical treatments and GT scoring followed Vu and coworkers (2020) since the scoring total GT callus number provides richer data than simply scoring GT cotyledon number.
During the GT reaction performed using the SpCas9-based pTC217 vector, treatment with the DNA ligase IV inhibitor led to an improvement in the GT e ciency compared to the mock control ( Figure 1a, upper panel). The GT e ciency was increased to around 3.3% with the treatments of either 10 or 50 µM SCR7 compared to the mock 0 µM treatment or the 1 µM SCR7 treatment (Supplemental Table 1). However, Fisher's LSD test for comparison of the GT e ciency between the treated concentrations and the mock control did not return signi cant p-values to determine whether the GT enhancement was strong enough (Figure 1a,upper panel). The data indicate that 10 µM SCR7 was the best concentration for SpCas9based GT in tomato. By contrast, the LbCas12a-based pHR01 construct exhibits a decreasing trend from the mock control to the highest SCR7 concentration. There were mild GT e ciency changes among the mock and 1 or 10 µM SCR7 treatments. Nevertheless, the GT e ciency was dramatically reduced to 4.140 ± 0.66% at 50 µM SCR7 from 6.95 ± 0.91% in the mock treatment (68% reduction) (Figure 1a, bottom panel; Supplemental Table 1).
To test the impacts of NU7441 and KU0060648 on CRISPR/Cas-based GT in tomato, we employed both single and multiple replicon systems to carry the SpCas9 or LbCas12a constructs. In the case of SpCas9, the GT e ciency was slightly increased (Figure 1b, top panel) when 0.2 µM KU0060648 or 1 µM NU7441, which were within the optimal concentration ranges tested in human cells (Robert et al., 2015), was applied to NSEL medium (Supplemental Figure 1) and incubated from days 3 to 8 post-transformation. However, pairwise comparison resulted in p-values that were not small enough to reject the null hypothesis. A similar situation also occurred for the LbCas12a-based single replicon, although NU7441 treatment led to a smaller p-value. In this case, 1 µM NU7441 treatment resulted in GT e ciency at 11.07 ± 1.50% compared to 7.17 ± 1.67% of that of the mock control, which represented a 1.54-fold enhancement ( Figure 1b, middle panel, Supplemental Table 2). The effect is considerably more clear with NU7441 treatment using the LbCas12a-based multiple replicon system. Blocking cNHEJ with 1 µM NU7441 signi cantly increased the multireplicon-based GT e ciency from 11.01 ± 0.93% in the mock control to 15.89 ± 1.15%, representing an approximately 1.44-fold change (Figure 1b, bottom panel, Supplemental Table 2). There was almost no GT e ciency change in the case of KU0060648 treatment with pMR01.
Silver nitrate treatment enhances GT e ciency and purple shoot regeneration Recently, polyamines, such as putrescine, spermidine, and spermine, were shown to enhance HDR by facilitating RAD51-mediated homologous strand annealing and synaptic complex formation in mice . We added 1 mM putrescine, spermidine, or spermine to the NSEL medium and incubated the transformed explants for 5 days after cocultivation with agrobacteria carrying the pMR01 plasmid. In contrast to the observations made in animals, no improvement in GT e ciency was achieved by any of the polyamines in the two replicates (Supplemental Table 3). In fact, the GT e ciency was reduced with supplementation with longer chain polyamines (i.e., spermidine and spermine) (Supplemental Figure 2). We also observed a higher proliferation of the purple GT calli and a delay in shoot formation from the explants treated with spermidine or spermine compared to the mock control. We surmised that direct supplementation with synthetic polyamines, especially spermidine and spermine, at high concentrations might not facilitate organ regeneration. Thus, we next used silver nitrate (AgNO 3 ) to increase endogenous polyamines. Silver nitrate was shown to stimulate the activity of arginine decarboxylase (ADC; EC 4.1.1.9), one of the key enzymes of putrescine and polyamine biosynthesis (Hanfrey et al., 2001). Using the pMR01 vector for this experiment, we treated the transformed explants with 30 µM AgNO 3 for 5 days on NSEL medium. In two replicates, we observed a signi cant increase in the number of purple GT spots per explant compared to that of the mock control at 21-day post-transformation (dpt) (Supplemental Figure 3a; Supplemental Table 4). Furthermore, the addition of AgNO 3 at a later stage, in the rst subculture with the SEL4C medium, stimulated shoot regeneration from purple calli (Supplemental Figure  3b). This nding is consistent with that silver nitrate enhances somatic embryogenesis ( Beyer, 1976;Giridhar et al., 2004;Roustan et al., 1990).
LbCas12a-based GT is superior to the SpCas9-based GT In our previous report, LbCas12a was shown to mediate GT more effectively than the SpCas9 system. The comparisons were conducted at the SlANT1 locus under various experimental conditions (Vu et al., 2020). However, the comparison using the replicon-based pTC217 and pHR01 constructs might have awed parameters, such as slightly different SSN-mediated binding and cutting sites and donors. Furthermore, the promoter and terminator driving the expression of SpCas9 and LbCas12a might also contribute to the differences. To better characterize and compare the GT performance of SpCas9 and LbCas12a, we designed a GT system with speci c SlANT1 cutting sites that are accessible to both nucleases ( Figure 2a and Supplemental Figure 4) and the same promoter and terminator to drive the transcription of Cas nucleases. The comparisons were conducted using single sgRNAs (sgR2 ANT1 vs. crR1.23 ANT1 and crR3.20 ANT1 , sgR3 ANT1 vs. crR3.23 ANT1 ) (Figure 2b and Supplemental Data S1 le). The 20-nt cRNA was also used for the LbCas12a (crR3.20 ANT1 ) to mimic the exact binding sequences of the SpCas9 gRNAs (Supplemental Figure 4). Each of the GT tools was expressed from both the replicon and T-DNA to compare the GT e ciency of the two delivery methods ( Figure 2b). Our data demonstrate the superiority of the replicon system compared to the T-DNA in plant GT, as the GT e ciencies were enhanced 5-8-fold with the replicons compared to that of the T-DNA tools ( Figure 2c and Supplemental Figure 5).
Moreover, in keeping with our previous data (Vu et al., 2020), all of the LbCas12a-based GT constructs, except the 20-nt crRNA, exhibited signi cantly higher GT e ciencies than the SpCas9-based GT tools (Figure 2c, 8.00 ± 1.11% of pHRC01 vs. 5.1 ± 0.35% of pHRC04, 9.53 ± 0.63% of pHRC03 vs. 6.73 ± 0.52% of pHRC05). These results also indicate that the 23-nt crRNA LbCas12a mediated GT more effectively than the 20-nt crRNA (pHRC03 vs. pHRC02). Surprisingly, analysis of the indel mutation e ciency of the guide RNAs at the plant stage demonstrated a reverse correlation between the indel mutation e ciency and the GT e ciency at cutting site 1 and cutting site 2 (Figure 2d, pHRC01 vs. pHRC04 and pHRC03 vs. pHRC05, Supplemental Figure 5). This result indicates that a considerably stronger indel mutation activity may negatively affect the GT reactions. Since cNHEJ-mediated indel mutations are favored throughout the life cycle of the cells and HDR is limited to the S-G2 phases ( Van Vu et al., 2019), DNA DSBs that form at cell cycles other than S-G2 may lead to permanent modi cations at the cutting sites, resulting in the inhibition of any further cleavage at these sites in the HDR-favorable cell cycles. Our data con rmed the stronger activity of SpCas9 in generating DNA DSBs compared to that of LbCas12a in tomato. The highest indel mutation rate was 49.5% on average with sgR2 ANT1 (Supplemental Figure 5). Most of the mutation traces appear to be deletions (Supplemental Figure 6). In the experiment, we obtained GT events with a typical purple phenotype due to the overexpression of SlANT1 and the subsequent accumulation of anthocyanin in the plants (Supplemental Figure 7) ttLbCas12a-based GT e ciency might be better than that of the wild-type variant in the case of the dual crRNA system.
The cleavage activity of Cas nucleases was shown to be temperature-dependent, especially in the case of LbCas12a (Malzahn et al., 2019). Previously, we showed the temperature dependency of CRISPR/Casmediated GT in tomato (Vu et al., 2020). In that case, LbCas12a exhibited considerably better GT support at temperatures as high as 31 °C compared to that at 19 °C or 25 °C. This result partially explained why SpCas9 was superior in indel mutation formation compared to LbCas12a at RT. Recently, Puchta's group reported a temperature-tolerant LbCas12a variant that signi cantly enhanced indel mutation (Schindele and Puchta, 2020) and GT (Huang et al., 2021;Merker et al., 2020a) e cacy in plants. The group used 24nt single crRNAs that were released by ribozyme anking sequences. It is interesting to characterize and compare the ttLbCas12a nuclease using our replicon system and on somatic cells of tomato. Here, we used a multiplexed editing approach with a single array of crRNAs and the gRNA length may affect the processing activity of the LbCas12a (Zetsche et al., 2017). We investigated the impacts of single and dual cleavages using 20-or 23-nt crRNA on the GT performance of both the wild-type and ttLbCas12a variants at the well-characterized SlANT1 locus (Figure 3a; Supplemental Figure 4 and Data S1 le). To contribute to the comparison data, another dual crRNA (crR1-2.23 ANT1 ) was also tested with LbCas12a_gRNA2 (Supplemental Figure 4), which was previously used by Vu and coworkers (Vu et al., 2020). Our data collected from 6 biological replicates were processed and compared using uncorrected Fisher's LSD test. The statistical analysis demonstrated very mild changes in GT e ciency among the GT constructs using single crRNAs with both LbCas12a variants ( Figure 3b; pHRC11, pHRC12, and pHRC13 for LbCas12a; and pHRC17, pHRC18, and pHRC19 for ttLbCas12a). The ttLbCas12a-based dual crRNA constructs (pHRC20, pHRC21, and pHRC22) showed higher GT e ciencies than LbCas12a (pHRC14, pHRC15 and pHRC16, respectively), although the p-values were only close to 0.05 and signi cant ( Figure  3b). The highest difference in GT e ciency was found between ttLbCas12a-containing pHRC21 (9.74 ± 1.49%) and LbCas12a-containing pHRC15 (6.44 ± 0.75%) with crR1-3.20 ANT1 , exhibiting a 1.51-fold change (p = 0.07) (Figure 3b and c).
In parallel with this experiment, we utilized targeted deep sequencing to analyze the cleavage activity of the nucleases with each of the gRNAs (Figure 3c) at 10 dpt. Due to the large size (~0.3x0.3 cm) of the cotyledon explants used in the previous study, the majority of the cells were not in direct contact with the agrobacteria and thus were not all transformed. Therefore, we reduced the size of the cotyledon explant to ~0.1x0.3 cm (Supplemental Figure 8) to reduce the untransformed cell portion for assessing the editing e ciency by targeted deep sequencing. The data collected from two biological replicates showed strong enhancement of indel mutation e ciencies of a gRNA if it was used in a dual crRNA construct, regardless of the LbCas12a variants. The largest enhancement was from LbCas12a_gRNA1 (23 nt) in the single crRNA crR1.23 ANT1 -carrying pHR11 (0.05%) and the dual crRNA crR1-3.23 ANT1 -expressing pHRC16 (1.66%) in replicate 1, representing a 33.2-fold increase ( Figure 3c). The cleavage activity of ttLbCas12a was also higher than that of the WT variant when single cuts were used. For example, in the case of the crR1.23 ANT1 -expressing constructs, the indel mutation e ciency was 5.00-to 7.29-fold increased with ttLbCas12a (pHRC17) compared to LbCas12a (pHRC11) (Figure 3c). For the cases of the dual crRNAs, ttLbCas12a showed more balanced indel mutation e ciencies between the two gRNAs used in the same construct, especially when the dual crRNA crR1-3.20 ANT1 with 20-nt gRNAs was examined (Figure 3c, pHRC21, and pHRC22 compared to pHRC15, and pHRC16, respectively). This activity might be one of the reasons that better enhancement of GT e ciency was mediated by ttLbCas12a compared to the WT version.
GT e ciency calculated by GT deep sequencing reads only showed precise gene editing at both loci in cases of dual crRNAs, although at low e ciencies (Figure 4c). No GT read was obtained from the replicon-based single crRNA constructs and the T-DNA-based dual crRNA regardless of the LbCas12a variants. At the SlHKT1;2 locus, the GT e ciency was higher for ttLbCas12a using the dual 20-nt crRNA (crR1-2.20 HKT1;2 ), but contrasting results were obtained with the longer dual crRNA (crR1-2.23 HKT1;2 ) ( Figure 4c). However, we obtained higher GT e ciency with the 23-nt dual crRNA (crR1-2.23 EPSPS1 ) for the replacement of the TIPS allele using ttLbCas12a (Figure 4c). These data indicate that the targeted deep sequencing method may help to identify the GT reads among the constructs, although the read numbers were still too low to be used for statistical comparisons. The indel mutation e ciency obtained from the T-DNA construct was up to 15-fold lower than that of the replicon using the same crR1-2.20 HKT1;2 (Figure 4c). In this experiment, the indel mutation e ciencies of the single crRNAs were also lower than those of the dual crRNAs. The highest indel mutation activity was obtained with crR1-2.20 HKT1;2 and LbCas12a, yielding values of 4.32% of LbCas12a_gRNA1 and 2.56% of LbCas12a_gRNA2. The gap between the indel mutation e ciencies of LbCas12a_gRNA1 and LbCas12a_gRNA2 from crR1-2.20 HKT1;2 was also lower in the case of ttLbCas12a. However, ttLbCas12a did not perform well when it was expressed with the 23-nt dual crRNA (crR1-2.23 HKT1;2 ); hence, no GT read was obtained at 10 dpt using the combination (Figure 4c). In general, between the two replicates, higher indel activity in a construct was correlated with higher GT e ciency.
Additional experiments for the exchange of the GAAT allele using the 23-nt dual crRNAs with two (crR1-3.23 EPSPS1 ) or four cleavage sites (crR1-2.23 EPSPS1 and crR2-4.23 EPSPS1 ) and the LbCas12a variants (Supplemental Figs. 9-10) demonstrated that ttLbCas12a performed better in either case. In triplicate, the highest GT e ciency was obtained with ttLbCas12a-expressing constructs (pHRES2.11 compared to pHRES2.9) using the dual crRNA crR1-3.23 EPSPS1 , up to 0.015 ± 0.015% for G177A and 0.018 ± 0.009% for A268T (Figure 5a). Notably, the four crRNA constructs (pHRES2.10 and pHRES2.12) showed a mild reduction in the gRNA1 and gRNA3 indel mutation e ciencies, which were expected to be higher than those of the dual gRNA-expressing constructs due to the synergistic effects of two close cleavage sites ( Figure 5a).
To further validate the performance of the LbCas12a variants, we screened and analyzed transformants obtained from the transformation of the LbCas12a-and ttLbCas12a-based constructs for the exchange of the TIPS allele. CAPS screening by BpiI digestion of the PCR products anking SlEPSPS1-targeted site 1 demonstrated potential TIPS allele-carrying events ( Figure 5b). The BpiI site located 84 bp downstream of the T178 codon was modi ed during the cloning of the homologous DNA donor through the Golden gate cloning method. Therefore, the undigested bands that appeared on the agarose gel were potentially derived from the TIPS-carrying alleles. Using the PCR products of the events that showed undigested bands in the CAPS assay for targeted deep sequencing analysis revealed several transformants carrying the precise GT allele at various frequencies and up to 9.003% in the case of the H281.82 plant generated with the ttLbCas12a construct (Table 1). Interestingly, the GT frequency revealed from ICE Synthego decomposition of the Sanger data of the same event showed only at 7% (Supplemental Figure 11). Two of the transformants (#H281.50 and #H281.82) obtained by the ttLbCas12a-based construct show targeted deep sequencing GT frequency at more than 5% that may be potential for obtaining inherited offsprings (Table 1). Based on that threshold frequency, we revealed that only the ttLbCas12a construct resulted in 2.78% GT e ciency at the plant stage ( Figure 5c). Besides, Sanger sequencing of the puri ed PCR products from 77 and 72 transformants of the wt LbCas12a and ttLbCas12a variant, respectively, and analysis of their .ab1 les by ICE Synthego (Hsiau et al., 2019) identi ed similar indel mutation e ciencies induced by the Cas proteins (Figure 5d). The average indel mutation e ciency was 29.36 ± 2.84% with LbCas12a and a similar rates for ttLbCas12a (28.03 ± 3.24%) (p = 0.5389, Student's t-test). Notably, the SlEPSPS1 TIPS allele-carrying GT plants showed no phenotypic changes compared to the WT plant (Supplemental Figure 12, pHRES2.8 events #82). Several events that contained high indel mutation rates exhibited phenotypic defects compared to WT (Supplemental Figure 12, pHRES2.7 events #27 and 28). NU7441 treatment enhances the ttLbCas12a-base GT in tomato Previously, NU7441 exhibited enhancement of LbCas12a-based GT with SlANT1 as a visible marker (Figure 1b). To further optimize ttLbCas12a-based GT in tomato, we investigated the impacts of various NU7441 concentrations on the GT process using ttLbCas12a constructs with crR1-2.20 HKT12 . Targeted deep sequencing data demonstrated the enhancement of GT e ciency with 1 µM NU7441 added to the culture medium albeit at low read numbers. However, when the NU7441 concentrations were higher, the GT e ciency was lower than that of the mock control ( Figure 5e). Notably, the NU7441 treatments at various concentrations showed only mild changes in the indel mutation e ciency of gRNA1 and gRNA2 of the dual crRNA (Figure 5e).

Discussion
The introduction of DNA DSB(s) at the targeted sites was shown to dramatically enhance GT e ciency in plants (Puchta et al., 1996). DSB repair is dictated by the NHEJ mechanism due to the abundance of KU70/80 and the other components in the cells ( Van Vu et al., 2019). Therefore, the plant HDR e ciency is considerably lower than that of NHEJ. The HDR pathway is more strictly dependent on the cell cycle, and even during favorable S-G2 phases, HDR must also compete with NHEJ to repair DNA DSBs. Previously, accumulated data in animal and plant studies showed the possibility of regulating repair pathway determination or inhibiting cNHEJ components by biochemical or chemical approaches (Chu et al., 2015;Endo et al., 2016;Maruyama et al., 2015;Nishizawa-Yokoi et al., 2016;Robert et al., 2015;Song et al., 2016;Srivastava et al., 2012;Zhang et al., 2017). Among the chemicals that exhibited positive effects on HDR or GT in animals, we chose to study several chemicals that inhibit the cNHEJ component(s) to enhance GT e ciency in tomato.
DNA ligase IV was shown to be involved in the last step of cNHEJ-mediated DSB repair to seal the broken ends of DNA DSBs. In a cell-free system, human DNA ligase IV was inhibited by SCR7, a small molecule chemical, by blocking its DNA binding activity (Srivastava et al., 2012). SCR7 at a concentration of 1 µM was shown to enhance CRISPR/Cas9-based GT e ciency up to 5-fold (Chu et al., 2015) or 19-fold (Maruyama et al., 2015) in mammalian and mouse cells, and 10 µM or 60 µM SCR7 treatment led to reduced transfection e ciency and cell viability. However, our data demonstrate only a moderate increase in GT e ciency with the SpCas9 construct at toxic levels of SCR7 in animals and no signi cant enhancement of GT e ciency in the case of LbCas12a (Figure 1a; Supplemental Table 1). Because DNA ligase IV inhibition by SCR7 was irrespective of the DSB con guration (Srivastava et al., 2012), the DNA DSBs generated by either SpCas9 or LbCas12a were not expected to affect the inhibition strength. Since our improved LbCas12a-based GT system showed high e ciency under the experimental conditions, the addition of SCR7, which affects the nal step of cNHEJ, may not be recognized easily, and the impacts of its toxicity level may be more visible (Figure 1a). There were limited data regarding the uses of SCR7 for CRISPR/Cas-based GT enhancement in plants; therefore, it is unclear if the SCR7 treatment is speciesdependent, since the uptake of SCR7 may be different in plants. The enhancement impacts of SCR7 were also controversial in animals since studies that used human cell lines or rabbit embryos did not show signi cant improvement of CRISPR/Cas9-or TALEN-based GT e ciency by SCR7 (Gutschner et al., 2016;Song et al., 2016;Yang et al., 2016).
One of the alternative strategies for blocking the cNHEJ pathway is to inhibit the DNA-PKcs or the other DNA-dependent protein kinases of the PI-3K family by NU7441 (Leahy et al., 2004) or KU0060648 (Munck et al., 2012). Studies conducted on mammalian cells showed that CRISPR/Cas9-based GT e ciency is enhanced up to twofold by using NU7441 or KU0060648 (Robert et al., 2015). However, treatment with 1 µM NU7441 and 200 nM KU0060648 did not signi cantly enhance the SpCas9-based GT e ciency under our experimental conditions, although only NU7441 signi cantly enhanced the LbCas12a-based GT e ciency up to 1.51-fold in the case of the multireplicon construct (Figure 1b; Supplemental Table 2) thanks to its ability to supply the homologous donor templates by the smaller-sized replicons (Vu et al., 2020). The NU7441 treated sample transformed with the LbCas12a-based single replicon pHR01 showed a tendency of higher GT e ciency compared to the control with the p-value was closed to 0.05. Since no plant homologs of DNA-PKcs have been identi ed, these data were surprising but were within our expectations for the existence of other types of plant DNA-dependent protein kinases ( Van Vu et al., 2019). More importantly, these data indicate that the inhibitory effects of NU7441 and KU0060648 were selective for the kinase forms and the con gurations of DNA DSB ends. Notably, although KU0060648 showed a wider range of inhibition of both DNA-PKcs and PI-3K compared to NU7441, only NU7441 had a positive impact on the LbCas12a GT tools, indicating that the kinase targets of KU006648 and NU7441 in tomato were distinct.
To further improve GT systems, recent studies using polyamines  and silver nitrate, a polyamine biosynthesis regulator, were conducted. In a cell-free assay, polyamines were shown to facilitate RAD51 activities during the formation of synaptic complexes and strand invasion. Depletion of polyamines resulted in the impairment of HDR in mouse hair follicle cells. However, to the best of our knowledge, no direct evidence of the addition of polyamines exerting stimulatory effects on HDR has been reported. Although direct supplementation with polyamines did not affect the CRISPR/Cas-based GT e ciency under our experimental conditions, when we treated the explants with silver nitrate, indirect stimulation of polyamine production using silver nitrate increased GT events, which appeared as purple counting data at 21 dpt (Supplemental Figure 3a). In the regeneration stage, silver nitrate might also suppress the activities of ethylene released under the stress induced by agrobacteria and tissue culture processes, leading to the promotion of embryogenic callus proliferation and subsequent shoot regeneration (Supplemental Figure 3b) ( Beyer, 1976;Giridhar et al., 2004;Roustan et al., 1990).
Previously, we showed that our LbCas12a-based GT system-mediated GT more effectively than did SpCas9 complexes (Vu et al., 2020). However, direct evidence for the comparison is still required, considering that the accessibility of the cleavage sites and loci by CRISPR/Cas complexes may exhibit different results. Therefore, in this study, we selected the well-characterized SlANT1 locus and its two cleavage sites (1 and 3) to compare the two nucleases using the same or very closed gRNA binding sites (Figure 2a and Supplemental Figure 4). The only difference between the SpCas9-and LbCas12a-based vectors was the coding sequence of each of the nucleases, and they were both human-codon optimized (Figure 2b and Supplemental Data S1 le). As expected, the LbCas12-based GT tools outperformed the SpCas9-based replicons at the same cleavage sites (Figure 2c). Notably, assessment of indel mutation e ciencies of the two systems at the plant stage demonstrated a reverse correlation with the GT e ciencies, as the SpCas9-based indel mutation e ciencies were signi cantly higher than those of the LbCas12a complexes at the tested cleavage sites (Figure 2d and Supplemental Figure 5). We reason that the strong cleavage activities of the SpCas9 complexes at all the cell cycles may lead to inhibition of the cleavage sites for further recurrent cuts and hence a reduction in the probability of homologous DNA donor-mediated repair by GT in the S-G2 HDR favorable phases. Another important point is that LbCas12a cleaves the targeted sites at a distal side of the TTTV PAM; hence, recurrent cleavages may be possible if the DSB repair of the rst cut did not affect the seed sequence located at the proximal side of the PAM (Vu et al., 2020). The view was also supported by a recent study (Huang et al., 2021). We cannot exclude the possibility that the difference in the DNA DSB con gurations of SpCas9 (mostly blunt ends) and LbCas12a (cohesive ends) differentially determined repair pathway activation in a spatiotemporal manner, which resulted in the difference in GT e cacy of the two nucleases. In addition, this experiment also indicates that the T-DNA-based gene editing e ciencies were notably low compared to that of the replicon system (Figure 2c, d) at the same cleavage sites due to its low copy nature.
Recently, in attempts to further improve GT e ciency in plants, Puchta's group found that a single mutation (D156R) of LbCas12a signi cantly improved GT performance compared to WT nuclease (Huang et al., 2021;Merker et al., 2020b), especially at the optimal temperature for plant growth. However, a direct comparison of the LbCas12a variants in somatic cell systems has not been reported. Our data not only provide a direct comparison of the two nucleases but also showed their crRNA preference ( Figure  3) in tomato somatic cells for practical applications. There was no signi cant improvement in GT e ciency using single crRNAs, but we observed that the ttLbCas12a-based GT might be enhanced with the dual crRNAs although the p-value was not less than 0.05 (p=0.0714), especially with the 20-nt crRNAs at the SlANT1 locus (Figure 3c). The weak improvement in GT e ciency mediated by ttLbCas12a compared to the data published by Puchta's group (Huang et al., 2021;Merker et al., 2020a) could be explained by the high-temperature experimental conditions applied from day 3 to day 12 throughout our study (Supplemental Figure 1), which might reduce the low-temperature tolerance advantages of ttLbCas12a. The slight difference from the 20-nt crRNAs might also be explained by the potential trans activities of the LbCas12a-crRNA complexes without affecting the on-target activities (Figure 3c) (Fuchs et al., 2019). Further comparison of the LbCas12a variants at the other two loci, SlHKT1;2 and SlEPSPS1, without using a donor or GT allele-associated selection marker showed better performance of ttLbCas12a with dual 20-nt crRNAs at SlHKT12a (Figure 4) and dual 23-nt crRNAs at SlEPSPS1 loci (crR1-2.23 EPSPS1 and crRNA1-3.23 EPSPS1 ) (Figures 4 and 5a). The data demonstrated the importance of using two neighboring (Figure 4a and b) or distancing cleavages (Supplemental Figure 9) for e cient GT, as this approach might offer synergistic effects in the case of neighboring cleavages that lead to considerably higher cutting . In the case of GT alleles that require two distanced sequence modi cations, such as the GAAT allele of the SlEPSPS1 loci, two DSBs anking the targeted sites may ensure simultaneous exchanges of the sequences, since the HDR-based conversion tract generated from each targeted site may not cover the other, due to length limitations (Huang et al., 2021).
Analysis of the transformants obtained from the GT experiment for exchanging the TIPS allele of SlEPSPS1 without using an allele-associated selection marker (i.e. glyphosate herbicide) revealed high GT e ciency (2.78 %) at the plant stage only with the ttLbCas12a-based construct (Figure 5c, Table 1).
The number of analyzed plants might not be su ciently high to obtain transformants with more than 5 % GT frequency in the case of the LbCas12a-based construct. The transformants carrying up to 9.003 % of the GT allele (Table 1) were found with normal phenotypes compared to WT parental plants (Supplemental Figure 12). Nonetheless, it would be important to evaluate the inheritance of the GT allele in the next generations, which was also shown in our previous report with the SlHKT1;2 N217D GT allele using the similar replicon-LbCas12a combination (Vu et al., 2020). We also observed abnormal phenotypes of the transformants carrying high indel mutation rates, especially those obtained from the LbCas12a-based construct. It was due to the high rates of SlEPSPS1 indel mutation alleles present in the same event. The malfunction of the SlEPSPS1 protein might lead to the ine ciency of aromatic amino acid biosynthesis, which causes phenotypic defects. Finally, we con rmed the stimulating impacts of 1 µM NU7441 on ttLbCas12a-based GT performance. However, due to the limited number of the targeted deep sequencing reads that carried the GT allele, even with the thin cotyledon explants, the potentially positive roles of NU7441 in plant GT may be further con rmed by targeted deep sequencing using a protoplast system. Considering that only the ttLbCas12a resulted in events carrying moderate TIPS GT frequencies among fewer than a hundred analyzed plants without using any allele-associated selection marker, ttLbCas12a is a better choice for plant GT. Our ndings from this work may add more or less positive information for the improvement of the plant GT system and may further facilitate its practical applications for precision crop breeding.

Conclusion
The natural HDR e ciency in plant somatic cells is too low to be utilized for practical applications of GTmediated plant breeding. Continuous efforts to improve GT performance have been undertaken for precision crop breeding. In this study, we further improved the LbCas12a-based GT system with the use of chemical treatments (1 µM of NU7441 and/or AgNO3). The impacts of small molecule chemical treatments on GT have not been well studied in crop plants. Therefore, our data for the assessment of the effects of SCR7, NU7441, and KU0060648 treatments on GT e ciency may help to elucidate their impacts and possible targeted components for HDR pathway regulation in plants.
Our data show that LbCas12a outperformed SpCas9 under the same experimental conditions at the SlANT1 loci. Similar effects of the replicon system for GT are also clearly indicated. Despite the milder stimulatory effects on GT performance under our experimental conditions due to the high-temperature protocol, the results of this study indicate that ttLbCas12a is a better choice for future applications in practical GT in plants. Taken together, the combination of the replicon with ttLbCas12a, double cleavages anking the modi cation sequence, and the addition of NU7441 and/or AgNO3 and appropriate temperature conditions are important parameters for the application of GT in future practical applications in precision plant breeding.

Experimental Procedures
System design for plant GT in tomato The SlANT1, SlHKT1;2, and SlEPSPS1 loci were used to conduct HDR-based DNA insertion and allele replacement experiments. The chemical treatment experiments used a single replicon (pTC217 and pHR01) and multiple-replicon tool (pMR01) vectors from previous works (Cermak et al., 2015;Vu et al., 2020) to target the SlANT1 gene. The SpCas9 containing the pTC217 vector was ordered from Addgene (Plasmid #70018) (Cermak et al., 2015). Further works used the single replicon system as a vector for the delivery of guide RNA and CRISPR/Cas expression cassettes and GT donor templates in this study ( Figure 2a and Supplemental Data S1 le).
For comparison of the SpCas9 and LbCas12a complexes in GT performance, we designed an HDRmediated insertion of selection markers at the SlANT1 locus that was well studied in our laboratory (Vu et al., 2020). The gRNA binding and cutting sites were selected in a manner that could be used for both SpCas9 (SpCas9_gRNA1 and SpCas9_gRNA2) and LbCas12a (LbCas12a_gRNA1 and LbCas12a_gRNA3) ( Figure 2a). We used a single gRNA for each of the GT vectors with either SpCas9 or LbCas12a ( Figure  2b). At the cutting site of LbCas12a_gRNA3, two different gRNA lengths (20 and 23 nt) were tested ( Figure 2b). All the editing constructs in the experiment were delivered by Agrobacterium-mediated transformation of tomato cotyledon explants, and two sets of constructs were cloned: one set with the single geminiviral replicon system (Vu et al., 2020) for the ampli cation of homologous DNA donors and the other T-DNA set for comparison.
For the assessment and characterization of the activities of ttLbCas12a in GT in comparison with the wild-type version of LbCas12a, similar constructs were designed for targeting the SlANT1 locus with single LbCas12a crRNA expression cassettes. In addition, dual guide RNA expression cassettes combining LbCas12a_gRNA1 (20 and 23 nt) and LbCas12a_gRNA3 (20 and 23 nt) were also used for the GT experiments (Figure 3a and Supplemental Data S1 le).
A similar system for comparison of ttLbCas12a and wtLbCas12a was designed for targeting the SlHKT1;2 and SlEPSPS1 loci (Figure 4a and b). Single and dual cutting sites were used for SlHKT1;2 ( Figure 4a and Supplemental Data S1 le). The gRNA lengths (20 and 23 nt) were also evaluated at the same locus. For SlEPSPS1, only dual gRNAs 23 nt in length were assessed for GT with the LbCas12a variants (Figure 4 b and Supplemental Data S1 le).
In all the GT vectors, the expression of SpCas9 and LbCas12a variants was driven by a long CaMV 35S promoter that contains an intron (Trp1) at the 5'UTR. A copy of AtUBQ10 intron 1 was also inserted in the coding sequence of the LbCas12a variants (Supplemental Data S1 le), which was tested previously for GT experiments by Vu and coworkers (Vu et al., 2020). The crRNA and sgRNA expression cassettes were transcribed with the support of the core sequence of the AtU6 promoter (Nekrasov et al., 2013).

Agrobacterium-mediated tomato transformation and chemical treatments
Agrobacterium-mediated tomato transformation was conducted following our protocol (Supplemental Figure 1) published previously by Vu and coworkers (Vu et al., 2020). For the treatment of chemicals, the chemicals at the tested concentrations were added to the NSEL medium. The 30 µM of silver nitrate was also added to the rst subcultured SEL4C medium at the later stage for stimulating somatic embryogenesis. The total treatment time was 5 days (day 3 to day 8 post transformation (dpt)). For targeted deep sequencing, cotyledon samples were collected at 10 dpt.

Assessment of GT e ciency
For assessment of the GT e ciency at SlANT1, purple spot counting was conducted at 21 dpt, and purple plants were recorded at the hardening stage. The GT e ciency re ected by the purple spot numbers was calculated with normalization to the SlANT1 overexpression tool that was transformed in parallel with the GT tool. Our calculation method normalized the number of cells/calli that was edited (and hence carrying p35S-ANT1 expression cassette) by the HDR tools to the number of cells that were successfully transformed with a T-DNA harboring over-expression cassette of the ANT1 gene driven by p35S in the same experimental conditions. This calculation may re ect more actual HDR frequency regarding the editing at the cell levels. A similar calculation was used and accepted elsewhere (Puchta et al., 1993;Puchta et al., 1996). This calculation method was also explained in our previous report (Vu et al., 2020).
For assessment of the GT e ciency at the SlHKT1;2 and SlEPSPS1 loci, targeted deep sequencing was conducted with thin cotyledon explants collected at 10 dpt. Sanger sequencing was performed to screen and validate GT plants.

Targeted deep sequencing
Genomic DNAs were isolated from the cotyledon explants (30 explants per sample) or mature leaves (three distinct lea ets from three different young leaves per plant per sample) using the CTAB method.
The MiSeq sequencing service (MiniSeq TM System, Illumina, USA) was used. MiSeq samples were prepared in three PCRs according to the manufacturer's guidelines with genomic DNAs as templates for the rst PCR. The rst and second PCRs used primers listed in Supplemental Table 5, whereas the third PCRs were performed with the manufacturer's primers to assign sample IDs. The rst PCR primers were designed for binding to the upstream and downstream sequences from the homologous donor sequence junctions to avoid amplifying the donor DNA sequences. The second PCR primers were designed to amplify 150-180-bp anking the targeted base changes at the targeted sites. High-delity DNA Taq polymerase (Phusion, NEB, USA) was used for PCR. The MiSeq raw data FASTQ les were analyzed by the Cas-Analyzer tool (Park et al., 2016). The indel analysis window was set to 5 bases, with a comparison range covering both read ends. The GT e ciency was assessed using the corresponding donor sequence as the input HDR donor sequence.

Statistical analysis
All comparison experiments were conducted with at least 3 replicates, and data were recorded by purple spot counting, targeted deep sequencing, and plant event screening. Some of the experiments using targeted deep sequencing were conducted in two replicates. The editing data, statistical analysis, and plots were further processed by the MS Excel and GraphPad Prism programs and explained in detail in the legends of gures and/or tables. Pairwise comparison data were tested with Student's t-test with unequal variance and two-tailed parameters. Similar parameters were applied for multiple comparisons using Fisher's LSD test. A difference was considered to be signi cant when the statistical tests returned a p-value < 0.05.   were conducted using Fisher's LSD test, and the p-values are shown above the compared boxes. (c) Indel mutation rates induced by the Cas-crRNA complexes that were assessed at 10 dpt by targeted deep sequencing method. The GT e ciencies are also added for comparison. Figure 4 GT performance of the LbCas12a variants at the SlHKT1;2 and SlEPSPS1 loci. (a-b) Schematic diagrams describing the expected GT processes for exchanging the homologous DNA donor template with the genomic sequence at the SlHKT1;2 (a) and SlEPSPS1 (b) loci. The D217 coding sequence was added during the cloning of the HKTD1 donor for exchange with the N217 sequence of the genomic site. The lengths of homologous arms are shown. Two cutting sites (LbCas12a cutting sites 1 and 2) were planned to support the GT. The reverse and forward primers for amplifying the targeted sites by PCR are shown with black arrows. The I178 and S182 coding sequences were added during the cloning of the EPSPS1D3 donor for exchange with the T178 and P182 sequences of the genomic site. The lengths of homologous arms are shown. Two cutting sites (LbCas12a cutting sites 1 and 2) were used for the GT experiments.

Declarations
The reverse and forward primers for amplifying the targeted sites by PCR are shown with black arrows. In (b), LbCas12a cutting site 2 is set as position 1, and the other positions are calculated accordingly. The diagrams were drawn not to their actual scales. (c) The GT and indel mutation e ciencies were assessed by targeted deep sequencing. At the SlHKT1;2 locus, four different crRNAs (single crRNAs: crR1.20HKT12; crR2.20HKT12, and dual crRNAs: crR1-2.20HKT12; crR1.23HKT12) were used for comparison of the LbCas12a variants in GT performance. A T-DNA vector was also used for comparison with the replicon system. With the SlEPSPS1 gene, only one dual crRNA construct (crR1-2.23EPSPS1) was used with the two LbCas12a variants. Wt: wild-type LbCas12a; tt: ttLbCas12a.

Figure 5
Further assessment of GT performance of the LbCas12a variants at the SlEPSPS1 locus. (a) Assessment of GT e ciency by targeted deep sequencing with GT tools using two or four cutting sites with LbCas12a variants at the SlEPSPS1 locus. (b) Screening of potential GT events using the CAPS assay at the plant stage. Transformants of each LbCas12a variant were obtained from the transformation of the GT tool with the crR1-2.23EPSPS1 expression cassette and used for genomic DNA isolation and PCR ampli cation of the targeted site with the UPEPSPS1-F2 and DNEPSPS1-R1 primers. The PCR products were puri ed and digested by the BpiI restriction enzyme since the BpiI site near the targeted site was modi ed in the DNA donor sequence. The red arrows indicate potential GT bands. 1-15: Representative transformants obtained from the transformation using the GT construct containing LbCas12a and crR1-3.23EPSPS1. (c) TIPS GT e ciencies obtained with the wt LbCas12a and ttLbCas12a. All the puri ed PCR products of the transformants that showed undigested bands at WT band size (2314 bp) in the CAPS assay were analyzed by targeted deep sequencing. Only the transformants carrying more than 5% of the TIPS allele were counted as GT events. (d) Boxplots showing indel mutation e ciency induced by LbCas12a and ttLbCas12a. All the puri ed PCR products used in the CAPS assay were sequenced by the Sanger method, and the ab1 les were subsequently analyzed by ICE Synthego software to reveal the indel mutation and GT e ciencies. The indel mutation e ciencies of all the samples were statistically analyzed using Student's t-test (unpaired, two-tailed, uncorrected)