Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation.MLPHhomozygous mutant quail exhibited gray plumage, whereasMLPHheterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions

Targeted gene knockout in Coprinopsis cinerea, yeast in vivo bioconversion and in vitro assays elucidated the lagopodin biosynthetic pathway, including a complexity-generating network of oxidation steps.

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

ABSTRACTStreptomyces iranensisHM 35 is an alternative rapamycin producer toStreptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation inS. rapamycinicus.The approach depends upon efficient conjugational plasmid transfer fromEscherichia colitoStreptomyces, and the failure of this step has frustrated its application toStreptomyces iranensisHM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions inS. iranensisHM 35. Among them wererapK, which generates the starter unit for rapamycin biosynthesis, andhutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants ofStreptomycesspecies that are genetically difficult to manipulate.IMPORTANCEThe developed protocol of conjugational plasmid transfer fromEscherichia colitoStreptomyces iranensismay allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable,Streptomycesspecies.