Dataset Information

Dataset on CRISPR/Cas9 system targeting hydrogenase genes in Rhodobacter johrii MAY2 isolate.

ABSTRACT: This dataset contains the gene sequences of the small and large sub-unit of the hydrogenase enzyme obtained from the annotated genome of Rhodobacter johrii MAY2. The whole genome sequence of the isolate was performed using SEED genome viewer on the Rapid Annotation using the Subsystem Technology (RAST) platform. Concurrently, guide RNA sequences and primers were meticulously crafted using the CHOPCHOP v.3.0 web tool, specifically designed for the precise editing and amplification of the target genes. The primers were optimized via gradient PCR to determine appropriate amplification conditions. Furthermore, the guide RNA was tested via in-vitro cleavage assay, gauging its efficacy in cleaving the intended target genes. The dataset, including the optimization and the cleavage assay, was deposited in Mendeley Data with DOI no: 10.17632/rcx3mcssnx.2.

SUBMITTER: Barcelo LAF

PROVIDER: S-EPMC10964040 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Dataset on CRISPR/Cas9 system targeting hydrogenase genes in <i>Rhodobacter johrii</i> MAY2 isolate.

Barcelo Leo Agustin F LAF Lantican Nacita B NB Manalang Aprill P AP Ventura Jey-R S JS

Data in brief 20240228

This dataset contains the gene sequences of the small and large sub-unit of the hydrogenase enzyme obtained from the annotated genome of <i>Rhodobacter johrii</i> MAY2. The whole genome sequence of the isolate was performed using SEED genome viewer on the Rapid Annotation using the Subsystem Technology (RAST) platform. Concurrently, guide RNA sequences and primers were meticulously crafted using the CHOPCHOP v.3.0 web tool, specifically designed for the precise editing and amplification of the t ...[more]

PMID: 38533121

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Project description:BackgroundGenome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies.ResultsWe sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used 'half-seed' explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75-100% mutation efficiencies. Indel frequencies varied widely among plants (3-100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27-100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants.ConclusionsUsing simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.

Dataset Information

Dataset on CRISPR/Cas9 system targeting hydrogenase genes in Rhodobacter johrii MAY2 isolate.

Publications

Dataset on CRISPR/Cas9 system targeting hydrogenase genes in <i>Rhodobacter johrii</i> MAY2 isolate.

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