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CRISPR/Cas9 mediated genetic resource for unknown kinase and phosphatase genes in Drosophila.

ABSTRACT: Kinases and phosphatases are crucial for cellular processes and animal development. Various sets of resources in Drosophila have contributed significantly to the identification of kinases, phosphatases and their regulators. However, there are still many kinases, phosphatases and associate genes with unknown functions in the Drosophila genome. In this study, we utilized a CRISPR/Cas9 strategy to generate stable mutants for these unknown kinases, phosphatases and associate factors in Drosophila. For all the 156 unknown gene loci, we totally obtained 385 mutant alleles of 105 candidates, with 18 failure due to low efficiency of selected gRNAs and other 33 failure due to few recovered F0, which indicated high probability of lethal genes. From all the 105 mutated genes, we observed 9 whose mutants were lethal and another 4 sterile, most of which with human orthologs referred in OMIM, representing their huge value for human disease research. Here, we deliver these mutants as an open resource for more interesting studies.

SUBMITTER: Wu M

PROVIDER: S-EPMC7193564 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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CRISPR/Cas9 mediated genetic resource for unknown kinase and phosphatase genes in Drosophila.

Wu Menghua M Zhang Xuedi X Wei Wei W Long Li L An Sainan S Gao Guanjun G

Scientific reports 20200430 1

Kinases and phosphatases are crucial for cellular processes and animal development. Various sets of resources in Drosophila have contributed significantly to the identification of kinases, phosphatases and their regulators. However, there are still many kinases, phosphatases and associate genes with unknown functions in the Drosophila genome. In this study, we utilized a CRISPR/Cas9 strategy to generate stable mutants for these unknown kinases, phosphatases and associate factors in Drosophila. F ...[more]

PMID: 32355295

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Project description:Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Project description:BackgroundThe plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants.ResultsIn the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean.ConclusionsOur results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

Dataset Information

CRISPR/Cas9 mediated genetic resource for unknown kinase and phosphatase genes in Drosophila.

Publications

CRISPR/Cas9 mediated genetic resource for unknown kinase and phosphatase genes in Drosophila.

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