We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource () for automated primer design. ![]() Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. ![]() ![]() CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids.
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