RESUMO
Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Genes de Plantas , Mutagênese/genética , Característica Quantitativa Herdável , Zea mays/genética , Sequência de Bases , Reparo do DNA/genética , Edição de Genes , Mutação/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos Testes , Moldes Genéticos , Transformação GenéticaRESUMO
The delivery of exogenous nucleic acids to eggs or non-embryonic individuals by microinjection is a vital reverse genetics technique used to determine gene function in mosquitoes. However, DNA delivery to eggs is complex and time-consuming, and conventional, non-embryonic-injection techniques may result in unobvious phenotypes caused by insufficient absorption of nucleic acid fragments by cells at target body parts or tissues. In this study, we developed a set of electroporation-mediated non-embryonic microinjections for the delivery of exogenous nucleic acids in Anopheles sinensis. Gene silencing using this method led to down-regulation of target gene expression (AsCPR128) by 77% in targeted body parts, compared with only 10% in non-targeted body parts, thus increasing the defect-phenotype rate in the target area by 5.3-fold, compared with non-shock injected controls. Electroporation-mediated somatic transgenesis resulted in stable phenotypic characteristics of the reporter gene at the shocked body parts during the pupal-adult stages in about 69% of individuals. Furthermore, injecting plasmid DNA near the ovaries of female mosquitoes after a blood meal followed by electric shock produced three germline G1 transgenic lines, with a transformation rate of about 11.1% (calculated from ovulatory G0 females). Among the positive G1 lines, 42%, 40%, and 31% of individuals emitted red fluorescence in the larval stage. When the red fluorescent larvae developed into adults, green fluorescence was emitted from the ovaries of the females upon feeding. These results suggest that electroporation-mediated non-embryonic microinjection can be an efficient, rapid, and simple technique for analyzing gene function in non-model mosquitoes or other small insects.