CRISPR/Cas9 has a potential to generate a targeting knockout in your research organisms

Masato Yoshizawa, Ph.D, Department of Biology, University of Hawaii at Manoa
Friday, March 13, 2015 - 3:30pm to 4:20pm
BioMed B-103

The genome editing technology is stepping in a new era. Traditionally, we have only a choice to make a targeting knockout using ‘mouse’, whose embryonic stem cell has relatively high efficiency to achieve homologous recombination between genome and introduced DNA sequence. Recently, zinc-finger nuclease technology was introduced as a novel targeting knockout method. This technology has a flexibility to design zinc-finger domain, therefore, to target specific sequence in the genome, and introduces double-strand-break in nearby genomic region. Due to incomplete DNA repair at the site of double-strand-break, it creates a null mutation--single or a couple of nucleotide deletion--at the targeted site, resulting in targeting knockout of gene function. The same idea was applied to more easy-to-use TALEN (more flexible design for DNA binding domain), and CRISPR/Cas9 (RNA-guided targeting) technologies.  These targeting knockout works in many organisms including yeast, Alabidopsis (plant), fruitfly, medaka, zebrafish, Xenopus, chicken, mouse, and human culture cells. In addition, they show substantially high efficiency to introduce mutations: for CRISPR/Cas9,  ~90% of cells carry mutation in a knockout animal. This afford us to investigate the gene function at the first generation without making further crossing (c.f. in knockout mouse, we need to cross twice to have a homozygote of gene knockout).  I have attended a workshop for TALEN, CRISPR technologies last year, and will discuss their flexibility and limit, in addition to a small advertisement of our research.