Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9） system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction（PCR）/restriction enzyme（RE） assay, T7 endonuclease I（T7EI） assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting（HRM） analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR（ACT-PCR）, for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

The advancements,challenges,and future implications of the CRISPR/Cas9 system in swine research

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(CRISPR/Cas9)genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds,thus improving yields.In addition,CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research.In this review,we present the advancements of the CRISPR/Cas9 system in swine research,such as animal breeding,vaccine development,xenotransplantation,and disease modeling.We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

C-terminal frameshift mutations generate viable knockout mutants with developmental defects for three essential protein kinases

Loss-of-function mutants are fundamental resources for gene function studies.However,it is difficult to generate viable and heritable knockout mutants for essential genes.Here,we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1(OsMPK1)results in weak mutants.This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations.Using the same C-terminal editing approach,we also obtained viable mutants for a wallassociated protein kinase(Os07g0493200)and a leucine-rich repeat receptor-like protein kinase(Os01g0239700),while the null mutations of these genes were lethal.These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus,which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering.