NHEJ deficiency develops homologous recombination in poplar

meaningfully further than the overexpression of HDR factors 2 3 Ali Movahedi, Hui Wei, Zhong-Hua Chen, Weibo Sun, Jiaxin Zhang, Dawei Li, Liming 4 Yang, and Qiang Zhuge 5 6 1 College of Biology and the Environment, Co-Innovation Center for Sustainable Forestry in 7 Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, 8 Nanjing Forestry University, Nanjing 210037, China 9 2 School of Science, Hawkesbury Institute for the Environment, Western Sydney University, 10 Penrith, NSW 2751, Australia 11 3 School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 12 210046, China 13 14 15 Correspondence should be addressed to Qiang Zhuge, Ali Movahedi, and Liming Yang: College 16 of Biology and the Environment, Co-Innovation Center for Sustainable Forestry in Southern 17 China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing 18 Forestry University, Nanjing 210037, China. E-mail: qzhuge@njfu.edu.cn; 19 ali_movahedi@njfu.edu.cn; yangliming@njfu.edu.cn; Fax: +86 25 85428701 20 21 § These authors are equally as the first author 22 23 24 Running title: XRCC4 deficiency improves HDR efficiency about 50 times in poplar 25

Introduction Precise, targeted genetic modification in trees has been challenging because of the 74 efficient NHEJ factors and also the difficulty of delivery of template DNA for HDR into the cell 75 nucleus. HDR has always been used to precisely repair DSBs, while NHEJs do the unexpected 76 and irregular repair in the DSB area. NHEJ has shown a predominant pathway and occurs among 77 the cell cycle widely (Panier & Boulton, 2014), while HDR rarely occurs (Puchta, 2005) because 78 of the low efficiency of transferring DNA or RNA fragments into the cell nucleus resulting in 79 difficulties to target and modify plant genomes accurately. Up to date, many studies have been 80 carried out to improve the genetic modification of crops by HDR. For instance, one study has 81 been carried out to increase ARGOS8 expression by replacing the GOS2 promoter via HDR to 82 drive ARGOS8 expression (Shi et al., 2017). Another report showed a ten-fold enhancement in 83 the efficiency of the insertion of the 35S promoter in upstream of the ANT1 gene in tomato 84 (Cermak et al., 2015). Some researchers also exhibited the promotion in gene-targeting  (Svitashev et al., 2015). 88 Although it has been previously reported using protoplasts for this purpose (Svitashev et   propagated under normal conditions. Vectors were then extracted using the plasmid midi kit 164 (Qiagen, USA) and confirmed by sanger sequencing (GenScript, Nanjing). 165 To produce DDT, we designed five fragments, constructed and ligated them, respectively 167 (Supplementary2a). To construct fragment one, the OsU3 promoter and gRNA scaffold were 168 isolated from pRGEB31 (Supplementary Table 2 To construct fragment three, we isolated the BleoR CDS from the PCR®-XL-Topo® 181 vector (Supplementary Table 2, BleoR-1092F and -2276R). Then, the overlap-PCR was 182 performed (Supplementary Table 2 Roche, USA) and performed three technical repeats for each event. Then, we used ANOVA-One 326 way to analyze the achieved mean data and compared. 327

RT-qPCR 328
We applied RT-qPCR using synthesized cDNA from grown buds on Zeocin (as 329 mentioned above) as the template and designed primers (Supplementary Table2, RT-qPCR part) 330 to investigate the expression of BleoR and MKK2 genes and their impact on each other. We also 331 explored our method's impact to develop HDR efficiency on HDR (CtIP, MRE11, BRCA1, 332 Rad50, and Rad51) and NHEJ (Lig4, XRCC4) influential factors. 333

Statistical analysis 334
All data were analyzed using ANOVA One-Way with Turkey means comparison 335

HDR strategy and target detection 340
Our purpose in this study was to increase HDR efficiency to integrate one exogenous into 341 the poplar genome. For this purpose, we decided to integrate BleoR exogenous with the 342 Supplementary 5d). We tried to transfer this plasmid into the plant cells with the same ratio of 393 4:1 pDDT/pggCtMR. In this experiment, recovered events were shown increased surprisingly to 394 twelve events from thirty-one grown buds on selection media. 395

Confirmation of transformants by western blotting, RT-PCR, and Southern blotting 396
Western blotting has been carried out to confirm the exact transformation to conjugate 397 the BleoR and MKK2 proteins. Using Western blotting, we also find out which grown buds on 398 Zeocin have been genetically edited by integrating the BleoR. We fused 6xHis tag sequences in the results, we did not observe any desired bond from ExI events. Then, we considered ExII and 415 Ex III events respectively and found out 3 (events #24, #29, and #35) and 4 (events #10, #23, 416 #36, and #45) 920 bp bonds (Figure 3e). Next, we considered the ExIV events and observed 9 417 (events #9, #17, #39, #45, #54, #60, #68, #72, and #83) increased desired bonds compared with 418 events included in ExII and ExIII. We then considered RT-PCR resulted from ExV and 419 surprisingly discovered 20 desired bonds. The second RT-PCR was also carried out, and results 420 revealed that we could not achieve the desired 413 bp bond (Figure 3f (Figure 4h). Our interpretation showed more 476 FAM signals remarkably measured in ExV than ExI, II, and-III. According to the analysis, we 477 detected the highest FAM signals from ExV events significantly more than ExI,-II, -III events 478 ( Figure 4h). Moreover, CtIP and MRE11 (ExIV) overexpression simultaneously increased these 479 signals and promoted HDR not as big as ExV events (Figure 4h). We also observed that the only 480 CtIP (ExII) or MRE11 (ExIII) were not able to improve HDR occurring significantly (Figure 4h). 481 The expression of exogenous BleoR integrated into the poplar genome was used as the 482 authority of HDR efficiency. We performed real-time PCR to evaluate the percentage of delta-483 delta Ct mean (Supplementary 15a) and then analyzed the mean achieved to compare and 484 illustrate the bar plot supported by standard distribution curves (Figure 4i; Supplementary 15b). 485 Our analysis revealed the BleoR expression a mean of -1.2287 from ExI events. This mean was 486 increased through EXII and EXIII events with 4.40787 and 6.11543. We then considered that 487 mean within EXIV events and discovered one increase of 19.06057. Finally, despite all our 488 previous observations, we found one significant development in BleoR expression integrated into 489 the poplar genome throughout EXV events of 48.90032. Our analysis proved this increase of 490 HDR efficiency significantly more than EXIV, EXIII, EXII, and EXI (Figure 4i). 491

The effect of efficient HDR on the expression of NHEJ and HDR factors 492
Regarding the new edition of the poplar genome in our study by integrating 493 exogenous BleoR fused with MKK2, we decided to investigate these two genes' expression and   (Figure 5f). 535 To test whether the promotion of HDR efficiency affects the happened polymorphisms, we 537 analyzed the polymorphism types, variants genotypes, protein effects, and nucleotide genotyping 538 of homology arms and also integrated BleoR into recovered events. We firstly analyzed the 539 homology arms for polymorphisms happened by HDR improvement experiments. Seven 540 polymorphism varieties including deletions, deletion tandem repeats, insertions, insertion tandem 541 repeats, SNP transitions with A to C or G to T and reversely, SNP transversions with Purines to 542 pyrimidines or reversely, and substitutions were detected through these editions (Supplementary 543 Table 3). We then analyzed all detected variant nucleotides together and found out that HDR 544 happening through ExI events induced the highest polymorphisms significantly more than ExIV 545 and -V events (Figure 6a). We also observed more happened polymorphisms through ExII and -546 III than ExIV and -V. Furthermore, happened polymorphisms through ExIV events were 547 observed more than ExV events (Figure 6a). 548 We then decided to investigate all polymorphisms in more detail on the homology arms and 549 the BleoR integrated into the recovered poplar genome (Supplementary Table 4 Moreover, the whisker plot of total polymorphisms presented the maximum distribution of 557 polymorphisms through ExI events and the minimum of those in ExV events (Figure 6b). 558

559
Despite extensive research on the use of HDR factors to increase HDR efficiency in 560 plants, no research has been reported using this system in haploid species such as poplar. We 561 hypothesized that if we increase the HDR pathway efficiency and decrease the NHEJ pathway 562 efficiency simultaneously, we might overcome it. To achieve this purpose, we must first increase 563 DDR amount in the cell nucleus correctly and at the right concentration. Therefore, we tried to 564 achieve this goal by increasing Agrobacterium concentration and increasing the plasmid ratio 565 containing DDT (pDDT) to plasmid containing gRNA (pgRNA) (Figure 2a). Based on the 566 we hypothesized that the grown buds were all transformed. Therefore, we investigated the grown 568 buds and transferred them to the rooting medium, including Zeocin and Cefotaxime, for recovery. 569 Many of the grown buds could not be recovered and died. 570 The western blotting was applied to verify the integration of the 6xHis tag fused with 571 exogenous BleoR. The observations proved the successful combination of BleoR through edited 572 events. All events that exposed 54.2 KDa were supposed to be successfully edited, and all events 573 that revealed 13.7 KDa were assumed might be unsuccessfully edited. For more details, we used 574 RT-PCR to investigate the precise integration of BleoR, followed by MKK2. For more confirmation, we used primers to probe BleoR applying southern blotting. We 580 discovered which events were edited successfully by our designed DDT regarding obtained 581 issues from western blotting, RT-PCR, and southern blotting. Because only edited events 582 integrated by BleoR or MKK2+BleoR could be recovered, therefore we figured out why some of 583 the grown buds on Zeocin were drowned and not recovered after transferring to rooting media. 584 For more investigation, we applied TaqMan real-time PCR for detecting designed FAM1 and 585 FAM2 signals (Figure 4b-g). Using this method, we compared the occurred HDR through all 586 grown buds involved in designed experiments by calculating the gained total FAM signals 587 ( Figure 4h). The highest total FAM from ExV events proved that XRCC4 deficiency caused 588 HDR development significantly more than overexpression of HDR factors. Moreover, the 589 investigation of HDR efficiency confirmed that the NHEJ pathway deficient is meaningfully 590 more efficient to HDR development compared to focus on only overexpression of HDR factors 591 (Figure 4i). 592 RT-qPCR and compared the BleoR and MKK2 expressions through all grown bud 593 transcriptomes helped us discover more deeply and accurately the role of decreasing activities of 594 the NHEJ and increasing activities of the HDR pathways in poplars. 595 The significant difference in the distribution of BleoR and MKK2 gene expressions within 596 ExI to -IV events confirmed that the overexpression of factors involved in the HDR pathway 597 20 alone could not have sufficient and appropriate ability to create homologous recombination in 598 the target genome. In contrast, the similar distribution of BleoR and MKK2 expressions through 599 ExV events proved that by reducing the NHEJ and improving the HDR pathway, we could 600 achieve appropriate and desired homologous recombination (Figure 5e). 601 We resulted in further Lig4 and XRCC4 expressions than the factors involved in the HDR 602 pathway (Figure 5f, ExI). Increase the CtIP expression within ExII and the MRE11 expression 603 within ExIII events caused to decrease the activities of XRCC4 and Lig4, but could not able to 604 significantly improve the other HDR factor activities and also to improve the development of 605 desired distributed expressions of BleoR and MKK2 (Figure 5b, c, and f, 5xII and III). The 606 simultaneously CtIP and MRE11 expressions caused to amplify the other HDR factor activities 607 and also to decrease the activities of XRCC4 and Lig4, but could not improve the development of 608 desired distributed expressions of BleoR and MKK2 (Figure 5d, and f; ExIV). 609 The XRCC4 deficiency caused not only to amplify the CtIP and MRE11 expressions 610 meaningfully but also caused a much more increase of the other HDR factor activities and a 611 much more decrease of Lig4 expression (Figure 5f, ExV). Furthermore, the XRCC4 deficiency 612 developed homologous recombinant and then desired distributed expressions 613 of BleoR and MKK2 (Figure 5e). 614 Moreover, we decreased the polymorphisms through XRCC4 deficiency significantly 615 compared with ExI (Figure 6a). XRCC4 deficiency also decreased in kinds of happening 616 polymorphisms compared to the overexpression of CtIP and MRE11 throughout the homologous 617 recombinant development (Figure 6a and b). Altogether, NHEJ deficiency caused to improve the 618 HDR efficiency in poplar meaningfully. 619 In summary, we have proved that XRCC4 deficiency can promote HDR, therefore greatly 620 expanding our capacity to improve hereditary developments in poplar. This breakthrough 621 technology is likely to encourage biotechnological researches, breeding programs, and forest 622 conservation of tree species. 623

Supplementary information 624
Supplemental information is available for this paper. 625  putatively edited events were regenerated on Zeocin. We allowed putative edited events to bud. 744 The elongated buds were then transferred on rooting media and allowed to be 745 recovered. Deletion and insertion occurred much more than the other types. SNP and substitution occurred 799 less than the other types. Whisker and standard distribution curves exposed that the total 800 polymorphisms caused by XRCC4 deficiency were less than the other experiments.   The overlap data are shown as bin bars, and the standard distribution curves are added. HDR efficiency plot revealed that XRCC4 deficient (ExV) let to HDR happening significantly more than the fusion of CtIP (ExII), MRE11 (ExIII), and CtIP+MRE11 (ExV). Also, ExIV meaningfully revealed more HDR happening than ExII and -III.; Error bars represent SE; Asterisks represent p-value as **≤0.01, ***≤0.001, and ****≤0.0001; Triplicate technical repeats were considered for each sample.   Whisker plot revealed that most polymorphisms happened in homology arms by ExI, and it was significantly more than those in ExV and -IV; Asterisks represent p-value as *≤0.05; Error bars represent SE. (b) Stacked column plot of total polymorphisms happened in DDT integration into the poplar genome. Deletion and insertion occurred much more than the other types. SNP and substitution occurred less than the other types. Whisker and standard distribution curves exposed that the total polymorphisms caused by XRCC4 deficiency were less than the other experiments.