Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing

Kazama, Tomohiko; Okuno, Miki; Watari, Yuta; Yanase, Shungo; Koizuka, Chie; Tsuruta, Yu; Sugaya, Hajime; Toyoda, Atsushi; Itoh, Takehiko; Tsutsumi, Nobuhiro; Toriyama, Kinya; Koizuka, Nobuya; Arimura, Shin‐ichi

doi:10.1038/s41477-019-0459-z

Cited by 134 publications

(117 citation statements)

References 43 publications

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“…Based on our data, we did not find evidence for NHEJ DNA repair in chloroplasts. This is consistent with prior reports in Chlamydomonas chloroplasts and in mitochondria of higher plants, showing the lack of NHEJ repair events (Odom et al, 2008;Kazama et al, 2019). In the latter, the engineered TALEN targeted to mitochondria was used to create a specific DNA cleavage.…”

Section: Cas9 Induced Homology-directed Repair Through Donor Dna Insesupporting

confidence: 91%

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Yoo¹,

Yadav²,

Orozco³

et al. 2020

PeerJ

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We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

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Section: Cas9 Induced Homology-directed Repair Through Donor Dna Insesupporting

confidence: 91%

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Yoo¹,

Yadav²,

Orozco³

et al. 2020

PeerJ

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“…The mechanism underlying the effects of orf463a on the CMS in the NWB CMS line and the interactions with nuclear genes are complex and need to be further characterized. Newly developed technologies, such as TALEN-mediated mitochondrial genome editing, may be useful for future investigations of CMS [43].…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial Genome Sequencing Reveals orf463a May Induce Male Sterility in NWB Cytoplasm of Radish

Wang

et al. 2020

Genes

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Radish (Raphanus sativus L.) is an important root vegetable worldwide. The development of F1 hybrids, which are extensively used for commercial radish production, relies on cytoplasmic male sterility (CMS). To identify candidate genes responsible for CMS in NWB cytoplasm, we sequenced the normal and NWB CMS radish mitochondrial genomes via next-generation sequencing. A comparative analysis revealed 18 syntenic blocks and 11 unique regions in the NWB CMS mitogenome. A detailed examination indicated that orf463a was the most likely causal factor for male sterility in NWB cytoplasm. Interestingly, orf463a was identical to orf463, which is responsible for CMS in Dongbu cytoplasmic and genic male sterility (DCGMS) radish. Moreover, only structural variations were detected between the NWB CMS and DCGMS mitochondrial genomes, with no nucleotide polymorphisms (SNPs) or meaningful indels. Further analyses revealed these two mitochondrial genomes are coexisting isomeric forms belonging to the same mitotype. orf463a was more highly expressed in flower buds than in vegetative organs and its expression was differentially regulated in the presence of restorer of fertility (Rf) genes. orf463a was confirmed to originate from Raphanus raphanistrum. In this study, we identified a candidate gene responsible for the CMS in NWB cytoplasm and clarified the relationship between NWB CMS and DCGMS.

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“…Molecular analysis of CMS has associated the S cytoplasm with mitochondrial open reading frames (ORFs) composed of pieces of duplicated genes and/or origin-unknown sequences [10][11][12], whereas other studies show associations with truncated mitochondrial genes, mitochondrial non-coding RNAs, and over-expression of mitochondrial genes [13][14][15]. Plants that have lost S cytoplasm-associated ORFs (hereafter S-orf s) from the mitochondrial genome have been obtained by mutation, somaclonal segregation or mitochondrial genome editing [10,16,17]. The phenotypes of these plants indicate that S-orf is responsible for male sterility but plays no other role in plant development.…”

Section: Introductionmentioning

confidence: 99%

What Does the Molecular Genetics of Different Types of Restorer-of-Fertility Genes Imply?

Kubo

Arakawa

Honma

et al. 2020

Plants

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Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production. Although male sterility is caused by S cytoplasm (male-sterility inducing mitochondria), the action of S cytoplasm is suppressed by restorer-of-fertility (Rf), a nuclear gene. Hence, the genetics of Rf has attained particular interest among plant breeders. The genetic model posits Rf diversity in which an Rf specifically suppresses the cognate S cytoplasm. Molecular analysis of Rf loci in plants has identified various genes; however, pentatricopeptide repeat (PPR) protein (a specific type of RNA-binding protein) is so prominent as the Rf-gene product that Rfs have been categorized into two classes, PPR and non-PPR. In contrast, several shared features between PPR- and some non-PPR Rfs are apparent, suggesting the possibility of another grouping. Our present focus is to group Rfs by molecular genetic classes other than the presence of PPRs. We propose three categories that define partially overlapping groups of Rfs: association with post-transcriptional regulation of mitochondrial gene expression, resistance gene-like copy number variation at the locus, and lack of a direct link to S-orf (a mitochondrial ORF associated with CMS). These groups appear to reflect their own evolutionary background and their mechanism of conferring S cytoplasm specificity.

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Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing

Cited by 134 publications

References 43 publications

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Mitochondrial Genome Sequencing Reveals orf463a May Induce Male Sterility in NWB Cytoplasm of Radish

What Does the Molecular Genetics of Different Types of Restorer-of-Fertility Genes Imply?

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