Team:HK SSC/Basic Part
Basic Parts
sgRNA
sgRNA, short for single guide RNA, is used in various CRISPR platforms. It acts as a guide for dCas9 (dead Cas9 enzyme) so that the enzyme specifically binds to the target sequence, and blocks transcription elongation by RNA polymerase[1]. It could act as a guide for CRISPR Cas9 enzymes, so that it specifically cleaves the target sequence.
McyB
McyB (microcystin biosynthesis cluster B) is a gene cluster responsible for Microcystin, a hepatotoxin, produced in Microcystis. It was found that repression of this cluster could lead no microcystin production. [2]
Design
The design of this sgRNA is according to Matthew H Larson[3]. It consists of a 25bp pairing region, a 42-nucleotide-long dCas9 handle hairpin for Cas9 protein binding, and a 40bp terminator. The base-pairing region targets the non-template strand of the McyB cluster of Microcystis Aeruginosa UTEX2388. It is unique throughout the genome of Microcystis Aeruginosa UTEX2388. The PAM site of this sgRNA is 5'CCN 3'.
We did not add promoters to this part, so that this part can be expressed in different hosts by cloning the part into a vector with suitable promoter (Promoters that are compatible in E.coli may not be compatible in Microcystis).
