Correction: Efficient CRISPR/Cas9-mediated genome editing of phytoene desaturase in Musa-AAA: a critical step for genetic improvement of East African highland bananas

Résumé

Banana (Musa spp.) is a perennial, herbaceous monocot cultivated both commercially and through subsistence farming across the wet tropics and sub-tropics (De Langhe et al., 2009). Edible banana varieties display diverse genomic constitutions, for instance, many sweet dessert and East African Highland bananas (EAHBs) have a triploid AAA genome, other cooking and starchy plantains and additional dessert bananas are AAB, ABB and AAAB while some seedless diploid (AA or AB) are also cultivated (Zorrilla-Fontanesi et al., 2020). The EAHBs which belong to the Lujugira-Mutika subgroup (with 'Lujugira' referring to a beer-type clone in Luganda) are especially vital to the food security of over 50 million people in the Great Lakes Region of Africa (GLA) (Kitavi et al., 2016).Banana productivity is greatly constrained by biotic factors such as pests and diseases, as well as abiotic factors like water, temperature, nutrient deficiencies and light intensity. These challenges have driven efforts to develop improved cultivars. In Uganda, scientists at the National Agricultural Research Organisation (NARO) have conventionally bred and released high yielding banana hybrids -NAROBan1, NAROBan2, NAROBan3, NAROBan4 and NAROBan5 with high resistance to black Sigatoka, a fungal disease caused by Mycosphaerella fijiensis that leads to substantial yield loses (Tumuhimbise et al., 2018). However, the triploid nature of EAHBs hampers the production of viable gametes which creates a significant barrier to introduce new germplasm through sexual recombination-based breeding methods (Kitavi et al., 2016).Advances in biotechnology such as marker-assisted breeding, genetic engineering, genome editing, synthetic biology, bioinformatics and systems biology offer promising strategies for developing banana varieties with enhanced traits of interest such as increased resistance to pests and diseases. For example, transgenic banana expressing rice Xa21 pattern recognition receptor has shown resistance to Xanthomonas vasicola pv. musacearum (Tripathi et al., 2014). However, the application of genetic engineering remains constrained by complex regulatory processes as well as persistent negative consumer perceptions. Meanwhile, the ever increasing availability of large biological data sets, sophisticated analytical tools and deeper understanding of biological systems has paved the way for innovative breeding approaches including genome editing.Genome editing employs site-specific endonucleases to introduce double-stranded breaks (DSBs) at precise target sites within the DNA, which are subsequently repaired through non-homologous end joining (NHEJ) or homology directed repair (HDR) mechanisms (Borrelli et al., 2018). Key genome editing technologies include clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (Mao et al., 2019). Among these, CRISPR/Cas9 genome editing has emerged as highly precise, efficient and versatile tool for genome editing across a wide range of dicotyledonous and monocotyledonous plant species (Bao et al., 2019;Hassan et al., 2021). Of the three CRISPR/Cas9 systems -type I, II, and III, the type II system derived from Streptococcus pyogenes is the most widely utilised. The type II system consists of two key components: the Cas9 endonuclease and a single guide RNA (sgRNA), which features a 20 nucleotide spacer sequence that directs the Cas9 protein to the target gene of interest, along with the a conserved Cas9 binding domain (Mali et al., 2013). With precise gene editing, this system introduces cuts in the DNA, allowing for the replacement, deletion, or insertion of specific sequences (Molinari et al., 2021).Phytoene desaturase (PDS) catalyzes the desaturation of phytoene to ζ-carotene which is then converted into lycopene and it interacts with multiple metabolites such as abscisic acid and strigolactones (Moise et al., 2014). The PDS gene has been used as a marker to successfully establish and confirm genome editing in a variety of plant species like Arabidopsis (Qin et al., 2007), apple (Nishitani et al., 2016), cassava (Odipio et al., 2017), melon (Hooghvorst et al., 2019a), strawberry (Wilson et al., 2019), rice (Banakar et al., 2020), papaya (Brewer & Chambers, 2022), celery (Liu, Li, et al., 2022a), chilli pepper (Bulle et al., 2024) and pigeon pea (Senthil et al., 2025).Despite their significance as a staple food in Uganda and across the Great Lakes region of Africa, there are currently no published reports on genome editing in EAHBs.Previous studies have demonstrated successful CRISPR/Cas9-mediated editing of the PDS gene in other banana cultivars, resulting in high rates of variegation; however, these outcomes suggest that editing efficiency may vary between cultivars (Ntui et al., 2020). This underscores the need to assess the feasibility of genome editing specifically in EAHBs. In the current study, we identified the PDS gene from the genome of the EAHB cultivar 'Nakitembe', which we sequenced in-house, and used it as a target to evaluate the efficiency and applicability of CRISPR/Cas9 in EAHBs. Our results provide a foundation for the precise genetic manipulation of key agronomic traits in EAHBs, with potential implications for improving food security in the GLA.The 4,006 bp sequence referred to as NktPDS was mined from the full genome sequence of the NKT and it was BLASTed against M. acuminata DH. Pahang (v4) genome using the Banana Genome Hub (https://banana-genome-hub.southgreen.fr/).Comparative analysis with M. acuminata DH Pahang (v2) revealed the gene model Ma08_t16510.2 had 100% identity, and it was therefore selected for exon mapping (Fig. 1). Ma08_t16510.2 had 14 exons and to maximise the likelihood of producing nonfunctional PDS transcripts, the first six exons were selected to create an intermediary sequence named "six NKT". To rule out SNPs, an alignment between NktPDS and "six NKT" was performed and the first 121 bp conserved region of NktPDS was identified and chosen to design the two sgRNAs. These sgRNAs were synthesised as oligonucleotide pairs (OP7/OP8 and OP9/OP10) with the appropriate adaptor sequences, as detailed in Supplementary information Table 1. Two sgRNAs were designed and individually cloned into the sgRNA expression plasmids pYPQ131C and pYPQ132C. These were then multiplexed into pYPQ142 via Golden Gate cloning. The resulting cassette was recombined with a Cas9 entry vector pYPQ167 and the binary vector pMDC32 to generate the final construct, pMDC32_Cas9_NktPDS (Fig. 2). The pMC32_Cas9_NktPDS was first transformed into E. coli DH5α for propagation and subsequently into A. tumefaciens strain AGL1 for banana transformation. Banana ECS lines NKT-732 and M30-885 were Agrobacterium-transformed with pMDC32_Cas9_NktPDS and pUBI:GUS, and sub-cultured on selective media for plant regeneration (Fig. 3). Histochemical GUS assays on cells transformed with pUBI:GUS construct showed blue-staining, confirming successful transformation of banana ECS.A total of 47 and 130 gene-edited events were regenerated for NKT and M30 respectively. Notably, the edited events exhibited slower growth compared to wild-type.More interestingly, M30 edited events began browning and wilting after two weeks on proliferation media, whereas browning in NKT was observed after one month, suggesting cultivar-specific physiological responses despite sharing the same genome group. As a result of browning, gene-edited events were frequently sub-cultured on proliferation media every month to maintain viability. A large number of these events were also kept in the dark to minimise photo-oxidation, thereby reducing oxidative damage and prolonging their survival in culture. phenotype (H, I) and wild-type control (J) on proliferation media.End-point PCR was performed to confirm the integration of Cas9 and hptII genes using primer pairs OP1/OP2 and OP3/OP4 respectively. All selected gene-edited lines from both NKT and M30 were confirmed positive for Cas9 and hptII genes, with amplicons of 560 and 398 bp respectively (Fig. 4). As expected, none of the wild-types tested positive for either gene. To enhance the precision of PDS gene editing, two sgRNAs spaced 719 bp apart were introduced into NKT and M30 cultivars. Gene-specific primers OP5 and OP6 were used in band-shift PCR to detect size differences between wild-type and gene-edited events.All regenerated events previously confirmed for Cas9 and hptII integration (Fig. 4) were selected and subjected to this analysis. In wild-type plants, primers OP5/OP6 amplify a 956 bp fragment. However, a successful dual-sgRNA-mediated deletion would result in a shortened amplicon of approximately 237 bp due to a 719 bp deletion.All the edited NKT events produced a 956 bp identical to the wild-type, suggesting only small indels (Fig. 5 A). Similarly, most M30 edited events showed a 956 bp band, except for events H2 and H17, which displayed an additional but smaller amplicon. Four gene edited M30 events (H2, H3, H17, H30), two gene-edited NKT events (N2, N25) and a wild-type were selected based on band shift PCR patterns and phenotype for mutational analysis by sequencing (Table 1). M30 events showed either deletions or insertions at sgRNA target sites. H2 showed a biallelic large deletion of 724 bp suggesting dual homozygous editing by both sgRNAs. H3 and H17 showed small deletions of 1 bp and 46 bp at gRNA2 (g2) and gRNA1 (g1) sites respectively suggesting homozygous edits while H30 showed both edited allele (+1 bp at g1) suggesting a heterozygous mutation. NKT lines showed various mutations with N2 displaying 98 bp deletion at g2 at one allele and small indels (+1/-2 bp; g1/g2) in another allele indicating a heterozygous mutation. N25 had a small deletion of 2 bp at g2 indicating a homozygous state. Both NKT and M30 events showed frameshift mutations, with only N2 and H2 showing mutations at both of the target sites. ----------------..719.. TGG--------------------- ----------------CAATGG ---------------- Note: WT; wild-type, "-"; deletion, light blue nucleotides; insertions and purple nucleotides; PAM.The regenerated gene-edited events exhibited various phenotypes, including completely white (albino) shoots, white shoots with green stripes (albino-variegated)and mixed green and white shoots (variegated), as illustrated in Fig. 6. In contrast, wildtype plants of both M30 and NKT exhibited uniform green shoots. Among the M30 gene-edited lines, 123 lines were albino, 6 were albino-variegated and 1 was variegated.All 47 gene-edited NKT lines were completely albino (Table 2). Notably, no nontransformed escape events were observed in either cultivar. The gene-edited events also exhibited dwarfism and, in some instances, produced very small shoots upon subsequent sub-culturing. A subset of both gene-edited and wild-type plants was selected for further analysis of carotenoid profiles and amounts. Ten gene-edited lines and one wild-type line per cultivar were selected for HPLC analysis based on band-shift PCR results and observed phenotypes. For NKT, the selected events included N-W, N1, N2, N3, N4, N5, N8, N9, N21, N25 and N31 while M30 were H-W, H2, H3, H9, H10, H11, H14, H17, H26, H30 and H31. HPLC analysis at 450 nm revealed that all gene-edited lines from both NKT and M30 lacked detectable levels of carotenoids, except for one variegated M30 event (H30) and one albinovariegated event (H31). In banana leaf tissue, three major carotenoid peaks -lutein, αcarotene and β-carotene were detected with retention times between 20 to 25 minutes.As expected, the M30 wild-type event (H-W) showed higher carotenoids than the variegated event (H30). Notably, in H31, lutein was the only carotenoid detected indicating a complete disruption of αand β-carotene biosynthesis and significant reduction in lutein production. Moreover, H-W had a higher total carotenoid content than the NKT wild-type (N-W), suggesting physiological differences between the two cultivars. The total carotenoid content, PVA carotenoids and BCE of selected NKT and M30 lines are summarised in Fig. 7 and Supplementary Table 2. albino-variegated. All data are presented as presented as mean± SD. Statistical analysis was performed using one way ANOVA revealing significant differences among groups (P=0.0001).Dunnett's multiple comparisons test indicated that both H30 and H31 had significantly lower carotenoid levels compared to the H-W at a significance level of P < 0.05 with adjusted Pvalues of 0.0004 and 0.0001 respectively.New plant breeding technologies like transgenesis, RNAi silencing, and genome editing offer transformative potential for crop improvement, including in banana. Genome editing, particularly using CRISPR/Cas9, introduces precise mutations similar to those or through is for efficiency and potential to crop The PDS gene has been used as a marker to establish CRISPR/Cas9 systems in various such as Arabidopsis (Qin et al., 2007), cassava (Odipio et al., 2017), melon (Hooghvorst et al., 2019a), strawberry (Wilson et al., and rice (Banakar et al., 2020). In the current study, the PDS gene was used to and evaluate the feasibility and efficiency of genome editing in East African Highland suggesting homozygous mutations, as in similar studies in celery (Liu, Li, et al., et al., blue et al., 2022), melon (Hooghvorst et al., and banana et al., of the gene-edited events revealed no detectable carotenoids in completely albino plants from both M30 and NKT cultivars. The albino-variegated M30 event only while the variegated event (H30) exhibited significantly carotenoid levels compared to the M30 wild-type (H-W) (Fig. are with studies such as et which showed that disruption of the PDS gene in leads to the wild-type NKT plants exhibited lower total carotenoids, β-carotene and A carotenoids compared to M30 wild-type plants (Fig. This with by et that total carotenoid content among from in to in acuminata cultivar with an of The observed differences between NKT and M30 may genetic the expression and of genes in carotenoid biosynthesis and this current study, PCR analysis using primers confirmed integration of Cas9 and hptII genes in all gene-edited lines, while wild-types showed no as (Fig. 4). PCR analysis revealed that all edited events in both cultivars produced an amplicon of 956 bp similar to wild-type with for event H2 and H17 (Fig. sequencing of selected gene-edited lines revealed frameshift mutations and a 100% efficiency in both frameshift mutations were for the disruption of the PDS gene of EAHBs to albino and variegated phenotypes. The gene-edited lines in the current displayed either insertions or deletions with the the revealed that of the gene-edited lines (H2, H3, H17, N25) in both cultivars had homozygous mutations while had heterozygous In contrast, et that of both and gene-edited events had homozygous results successful of the non-homologous end joining (NHEJ) repair with the large deletion in H2 confirming dual by the sgRNAs. The is a in plant genome editing, as it DNA, an for & Notably, a similar by et using two sgRNAs to target PDS in banana which to the genome to the band shift in edited This due to the that et used a Cas9 the expression of either a 1 or a single which leads to Cas9 as compared to the used in this current In the used a rice for sgRNA expression which may in as compared to The was to high sgRNA expression in rice than the et al., all studies have used in sequences of the PDS gene but the current used one derived from a full genome sequence of the the current study, both sgRNAs were in both cultivars with the most However, only two gene-edited lines and showed mutations at both sgRNA target sites. The differences in sgRNA efficiency at target sites factors including sgRNA such as the content, nucleotide and et al., et al., Cas9 on sites has been to sgRNA and efficiency et al., The by the complex to the target has been to sgRNA across species & 2021). of the complex at the target repair with editing outcomes (Liu, et al., The in sgRNA across cultivars has been in various plants such as pepper & and melon et al., The differences in editing efficiency of sgRNAs across cultivars due to genome differences among cultivars (Ntui et al., sequencing of H17 in this produced a 46 bp deletion the event displayed two from band shift This that the sequencing primer the wild-type like allele out the other allele with a deletion, which is a of PCR efficient sequencing approaches such as sequencing of cloned or sequencing are in PCR sequencing may mixed in the same the used in the current genomic which may for disruption of regulatory or of wild-type in most lines the high editing efficiency of the system used in the current gene-edited in this used banana transformation and are as plants in many regulatory This is construct such as and genes are into the plant genome which et al., in at are to develop approaches based on a transformation system the complex using the Cas9 protein and are into the plant genome are or after editing no in the genetic This leads to production of systems for plasmids and have been successfully in banana et al., et al., et al., 2020). However, regeneration of gene edited plants from banana remains a major plants have been produced in other for example, et al., and et al., The successful of a banana to plant regeneration system with the CRISPR/Cas9 system of improved EAHBs and dessert bananas with wide range of target the first successful of genome editing in East African Highland (EAHBs) including the cultivar NAROBan5 a significant in banana genetic As a of the CRISPR/Cas9 system demonstrated to large genomic deletions potential for gene in banana. This system a for gene and all of which are for developing improved EAHB EAHBs are a major staple and for in East and The to genes for developing East African banana cultivars with enhanced resistance to pests and diseases, improved and traits that the of farming of EAHB and NAROBan5 by and at the National Agricultural Research were used in the current A was to the end of and to the end of the of sgRNA as a for adaptor sequences and for the were to into using the The final including and along with their Table were synthesised by CRISPR/Cas9 construct, pMDC32_Cas9_NktPDS was to et the expression pYPQ131C and were using to bp The were using PCR to The and of the sgRNAs were and using The and sgRNA gRNA1 and were then into pYPQ131C and using to and respectively. The were transformed into E. coli strain selected on with 50 were selected and in from which was and for by with the were selected after which the two were into the Golden Gate and entry pYPQ142 by with and using The Golden Gate were also transformed into E. coli DH5α with of selected was and by PCR and using and A positive from the Golden Gate and the Cas9 entry vector were then cloned into the binary vector & in a using II The was then transformed in E. and cells were selected on and were by and using The of the final CRISPR/Cas9 binary vector gene as an in Cas9 gene and the two driven by the rice The Cas9 gene used in this is plant and is by pMDC32_Cas9_NktPDS was transformed into tumefaciens strain AGL1 as by et and transformed selected on with and were confirmed using of the of A. tumefaciens strain AGL1 with pMDC32_Cas9_NktPDS were for transformation of banana ECS while were and at for ECS lines of NKT and M30 were transformed with vector pMDC32_Cas9_NktPDS using a by et a single of A. tumefaciens pMDC32_Cas9_NktPDS binary vector was for in and of the pUBI:GUS construct gene the control of was similar For construct, a 5 was into 20 of and for the same of the was into 50 and at for at The was in 25 media with was for at and 25 and using A of banana cells were to 50 line into media was from the cells to which of media at was banana cells were in a at for 5 after which the media was With the of banana cells were in by of For the of media were Banana cells were two times at for and then at for of were on a and on media with were with in and at for and three times with media with of were to and in the dark on media for media was with and but no for control For of cells transformed with subsequently at in 50 of The and were confirmed by of Cas9 and hptII genes in the regenerated events was confirmed by primer OP1/OP2 and OP3/OP4 respectively. PCR of with for of the and of and of were in a with the at for 5 of for for 1 and for 1 by a final at for 7 then at PCR were then through a with mutations in the PDS gene were detected by band shift PCR analysis using the gene specific primers the two sgRNAs. The 50 PCR 1 of of and of PCR were the same as and PCR were through a with to a shift in size of the mutations at the target in the gene-edited plants, sequencing was performed on selected regenerated events previously confirmed by band-shift PCR using OP5 The PCR were and sequenced using the at the Research The sequences were against the wild-type PDS gene using to and regenerated were for various such as albino, and variegated which are due to the of the PDS as well as no variegated which is from type carotenoids were from 25 of leaf of wild-type and gene-edited the leaf was into a using a and was out by at 450 nm as by et was to leaf and for 5 at at The was and were and using and The was to the which was then and at in for analysis. carotenoids, A carotenoids and β-carotene were and as per of were performed in from the carotenoid were compared between gene-edited lines and lines with carotenoid were from as to were presented as analysis was performed using of by multiple test to assess significant differences between gene-edited lines and All were using with significance at

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