TY  - GEN
T1  - SapTrap vectors for introducing point mutations with unc-119+ selection
AU  - Schwartz, Matthew
AU  - Jorgensen, Erik
DO  - 10.17912/DDVH-BG64
UR  - http://beta.micropublication.org/journals/biology/ddvh-bg64/
AB  - SapTrap assembly generates CRISPR targeting vectors using a Golden Gate cloning strategy (Schwartz and Jorgensen, 2016). The original toolkit was designed for inserting tags, such as fluorescent proteins or affinity tags, into protein coding genes in C. elegans. In the original design, the floxed unc-119+ selectable marker was incorporated into the tag. For site-directed mutagenesis, the selectable marker is useful but a tag is unnecessary. We have developed a new set of vectors dedicated for introducing point mutations using unc-119+ selection. These new vectors contain only a floxed unc-119+ selectable marker in the reverse orientation of a synthetic intron (‘syntron’) that lacks both 5’ and 3’ splice sites. This unc-119+ module can be inserted directly into a native intron. Alternatively, splice site sequences can be appended for insertion into a coding sequence to generate a syntron in the coding sequence. The desired point mutations are designed into the homology arms of the selection construct. SapTrap is used to assemble a plasmid encoding the guide RNA and the template. After injection and selection of unc-119+ animals, the selectable marker can be removed by expression of Cre recombinase to generate an animal with only the desired point mutation and a single Lox site in the gene of interest.
PY  - 2018
JO  - microPublication Biology
ER  -