RESUMO
A novel promoter assay was developed for Agaricomycetes, using a gene-targeting approach, with or without the CRISPR/Cas9 technique. It enables precise evaluation of promoter activity at the original site of the chromosome without random and multiple integrations in conventional transformation experiments.
Assuntos
Agaricales/genética , Marcação de Genes , Pleurotus/genética , Regiões Promotoras Genéticas/genética , Sistemas CRISPR-Cas/genética , Celulase/genética , Regulação Fúngica da Expressão Gênica/genética , Peroxidases/genéticaRESUMO
A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.
