I study computational and quantitative biology with a focus on network aging. This site is to serve as my note-book and to effectively communicate with my students and collaborators. Every now and then, a blog may be of interests to other researchers or teachers. Views in this blog are my own. All rights of research results and findings on this blog are reserved. See also http://youtube.com/c/hongqin
Monday, June 18, 2018
CRISPR in budding yeast
Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.
To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.