Dataset Information

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens.

ABSTRACT: Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene knock-out-technology in a variety of species. However, in a number of situations knocking out or modifying a single gene is not sufficient, this is particularly true for genes belonging to a common family or for genes showing redundant functions. Like many plants the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the co-delivery of a CAS9 expressing cassette, multiple sgRNA vectors and a cassette for transient transformation selection for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9 mediated targeting of five different genes allows the selection of a quintuple mutant and all possible sub-combinations of mutants in one experiment with no mutations detected in potential off target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to alternative End Joining pathway for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

SUBMITTER: Lopez-Obando M

PROVIDER: S-EPMC5100863 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other

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Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in <i>Physcomitrella patens</i>.

Lopez-Obando Mauricio M Hoffmann Beate B Géry Carine C Guyon-Debast Anouchka A Téoulé Evelyne E Rameau Catherine C Bonhomme Sandrine S Nogué Fabien F

G3 (Bethesda, Md.) 20161108 11

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism <i>Physcomitrella patens</i> has experienced multiple events of pol ...[more]

PMID: 27613750

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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

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens.

Publications

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in <i>Physcomitrella patens</i>.

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