Promoter replacement by genome editing creates gain-of-function traits in Arabidopsis

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The resulting lines misexpressed FT and flowered early, like FT -overexpressing transgenic plants. This system can be used to create gain-of-function mutations that modify target gene expression as desired without incorporating foreign DNA sequences. Modifying target genes through genome editing technology has promising agricultural applications ( Wang et al., 2023 ). Typical genome editing employs site-directed nucleases (SDNs), such as clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9), to generate DNA double-strand breaks (DSBs) at specific sites in the genome, leading to DNA mutations caused by errors during repair. The mutations generated by genome editing using SDNs can be classified into three categories ( Matres et al., 2021 ). SDN-1 mutations are typically short deletions or insertions, arising through errors during DNA repair via non-homologous end-joining. SDN-2 and -3 mutations result from homology-directed repair, whereby foreign DNA sequences are introduced into the genome DNA. Even though genome-edited plants carrying SDN-1 type mutations were produced through transgenesis, they are not heavily regulated and are socially acceptable in many countries as long as their transgenes have been segregated out. Therefore, SDN-1 genome editing is useful for crop improvement, and many SDN-1 genome-edited strains have been developed. However, most SDN-1 genome-edited strains developed so far harbor loss-of-function mutations. Two DSBs on the same chromosome may cause a deletion or an inversion. Such inversions have been reported to be achieved in plants using CRISPR-Cas9 ( Schmidt et al., 2019 ). Because such modifications do not involve foreign DNA sequences, they are considered to be SDN-1 type mutations. Most induced inversions cause loss-of-function mutations. However, an inversion that creates a gene fusion involving the coding region of the target gene and the promoter of another gene may affect the expression of the target gene, leading to phenotypic changes. To test this possibility, we aimed to exchange the promoters of two genes on the same chromosome via targeted inversion in the model plant Arabidopsis ( Arabidopsis thaliana ), using CRISPR-Cas9 to introduce DSBs near the transcription start sites. We chose FLOWERING LOCUS T ( FT ) on chromosome 1 as the target gene. FT encodes florigen and leads to very early flowering when overexpressed ( Kardailsky et al., 1999 ). As a promoter donor, we used HTA3 , encoding a histone H2A variant. HTA3 is expressed in various young tissues, and its loss-of-function mutants exhibit normal growth ( Yi et al., 2006 ). HTA3 is also located on chromosome 1, about 3.6 Mb upstream of FT , with the two genes in divergent orientations. Thus, an inversion of the 3.6-Mb fragment between the two genes that contains their promoters would result in a promoter exchange, placing FT under the control of the HTA3 promoter. We therefore designed two single guide RNAs (sgRNAs) to introduce DSBs near the transcription start sites of FT and HTA3 ( Fig. 1a, b ). Download figure Open in new tab Figure 1. Targeted chromosomal inversion mediated by CRISPR-Cas9 confers an early flowering phenotype in Arabidopsis. (a) Diagram of targeted inversion between the FT and HTA3 loci. (b) Structures of the fusion sites between FT and HTA3 . Protospacer-adjacent motifs (PAMs) in sgRNAs are shaded in blue or green. Thin and thick boxes indicate untranslated regions (UTRs) and exons, respectively. (c) Strategy for screening for the HTA3-FT inversion using multiplex PCR-based genotyping. An inversion-positive individual is indicated in red. (d) and (e) , PCR-based screening for the HTA3-FT inversion using genomic DNA of bulked T 1 individuals (d) and individuals from the PCR-positive pool (e) . HTA-F1, HTA-R1, and FT-R1 primers (See panel A) were used for multiplex PCR. White and yellow arrowheads indicate the wild-type band and the band expected for the inversion, respectively; * indicates an inversion-positive sample. (f) Representative photograph of flowering in Col-0 and inversion segregants grown under short-day conditions at 36 days after germination (DAG). (g) Bolting time of transgene-free population segregating for the inversion under short-day conditions. (h) FT expression levels in Col-0 and inversion homozygotes. Total RNA was extracted from seedlings collected at 9 DAG under short-day conditions. ***, P<0.001 ( n =4). (i) Distribution of bolting time in the transgene-free HTA3-FT inversion line and the CaMV35S:FT line grown under short-day conditions. (j) Fresh weight was measured from the ten 14-day-old plants. n.s., not significant ( n =3). Statistical analyses were conducted using Student’s t -test. In this strategy, the HTA3 promoter should be joined to the FT coding region (H-F junction), and the FT promoter to the HTA3 coding region (F-H junction). We employed multiplex genotyping PCR that detects both the H-F fusion structure and the F-F wild-type structure ( Fig. 1a, c, d ). PCR genotyping of bulked DNA samples from 170 independent T 1 transgenic lines identified the H-F fusion in one bulked DNA sample containing six individuals ( Fig. 1d ). One of the six individuals showed a positive signal for the H-F fusion ( Fig. 1e ). We collected T 2 seeds from this T 1 plant, genotyped 14 T 2 plants for the H-F fusion, and examined their flowering time when grown under short-day conditions (Figure S1a). All individuals with at least one copy of the H-F fusion flowered early, whereas individuals that lacked the H-F fusion flowered normally. Thus, the H-F fusion was inherited by the progeny and is associated with early flowering. Sequencing of the PCR fragment derived from the H-F fusion confirmed that the HAT3 promoter was fused with the FT coding region, together with a 9-bp deletion ( Figure 1b ). For the F-H fusion, sequencing of the corresponding PCR amplicon detected a precise fusion between the FT promoter and HTA3 coding region at the intended break point positions. These findings indicated that an inversion of the 3.6-Mb genomic fragment between FT and HTA3 occurred in this individual. We generated a CRISPR-Cas9 transgene-free population segregating for the inversion by crossing a plant heterozygous for the inversion with Col-0. We observed a perfect one-to-one correspondence between the presence of an inversion and early flowering, although the flowering phenotype behaved as a semidominant trait ( Figure 1f, 1g ). The Col-0 plants transformed with a genomic fragment encompassing the inversion exhibited an early flowering phenotype, confirming that the inversion led to earlier flowering (Figure S1b). FT is not induced in the wild-type under short-day conditions, but was expressed at high levels in plants homozygous for the inversion under these conditions ( Figure 1h ). The misexpression of FT may therefore be responsible for the early-flowering phenotype of the plants harboring the inversion. RT-PCR indicated that the FT transcripts produced in the inversion line comprise their entire respective coding sequences (Figure S2). When grown under short-day conditions, all transgene-free F 6 plants homozygous for the inversion flowered as early as transformants overexpressing FT from the cauliflower mosaic virus (CaMV) 35S promoter (T 5 individuals homozygous for a single-locus transgene) ( Figure 1i ). While the inversion line exhibited a stably inherited early-flowering phenotype, the flowering time of CaMV35S:FT plants was more variable with some flowering as late as Col-0, suggesting possible silencing of the transgene. The growth and fertility of the transgene-free line with the inversion were comparable to those of the wild-type ( Figure 1j , Figure S3). These observations suggest that the genome-editing inversion system presented here can introduce gain-of-function mutations that confer stable agriculturally important traits, without affecting other traits. In this study, we successfully modified the expression of a target gene using SDN-1 genome editing in Arabidopsis, a feat that has previously been achieved primarily through creation of genetically modified organisms. This system potentially has interesting applications. For instance, a herbicide-resistant rice strain was developed through overexpression of a target of herbicide by CRISPR-Cas9-mediated inversion ( Lu et al., 2021 ). Beyond overexpression, modifying tissue specificity or enabling the inducible expression of a gene of interest could be achieved by selecting an appropriate promoter. Development of additional technologies, including induction of translocations ( Beying et al., 2020 ), would expand the applicability of this system by enhancing the availability of promoters. Furthermore, the recently reported Bridge RNA system, which achieved programmed recombination in Escherichia coli , could be incorporated into this system for the innovative crop improvement ( Durrant et al., 2024 ). References ↵ Wang JY , and Doudna JA ( 2023 ) CRISPR technology: A decade of genome editing is only the beginning . Science 379 : eadd8643 . OpenUrl CrossRef PubMed ↵ Matres JM , Hilscher JH , Datta A , Armario-Nájera V , Baysal C , He W , Huang X et al. ( 2021 ) Genome editing in cereal crops: an overview . Transgenic Res . 30 : 461 – 498 . OpenUrl PubMed ↵ Schmidt C , Pacher M , and Puchta H ( 2019 ) Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system . Plant J . 98 : 577 – 589 . OpenUrl CrossRef PubMed ↵ Kardailsky I , Shukla VK , Ahn JH , Dagenais N , Christensen SK , Nguyen JT , Chory J et al. ( 1999 ) Activation tagging of the floral inducer FT . Science 286 : 1962 – 1965 . OpenUrl Abstract / FREE Full Text ↵ Yi H , Sardesai N , Fujinuma T , Chan C-W , Veena, Gelvin SG ( 2006 ) Constitutive Expression Exposes Functional Redundancy between the Arabidopsis Histone H2A Gene HTA1 and Other H2A Gene Family Members . Plant Cell 18 : 1575 – 1589 . OpenUrl Abstract / FREE Full Text ↵ Lu Y , Wang J , Chen B , Mo S , Lian L , Luo Y , Ding D et al. ( 2021 ) A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice . Nature Plants 7 : 1445 – 1452 OpenUrl PubMed ↵ Beying N , Schmidt C , Pacher M , Houben A , and Puchta H , ( 2020 ) CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis . Nature Plants 6 : 638 – 645 . OpenUrl CrossRef PubMed ↵ Durrant M , Perry NT , Pai JJ , Jangid PA , Athukoralage JS , Hiraizumi M , McSprdon JP et al. ( 2024 ) Bridge RNAs direct programmable recombination of target and donor DNA . Nature 630 : 984 – 993 . OpenUrl CrossRef PubMed Hiraizumi , M , Perry NT , Durrant MG , Soma T , Nagahata N , Okazaki S , Athukoralage JS et al. ( 2024 ) Structural mechanism of bridge RNA-guided recombination . Nature 630 : 994 – 1002 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted March 25, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Promoter replacement by genome editing creates gain-of-function traits in Arabidopsis Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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