Part:BBa_K3219001

sgRNA for mcyB Microcystis Aeruginosa UTEX 2388

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. It could act as a guide for CRISPR Cas9 enzymes, so that it specifically cleaves the target sequence.

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.

The PAM site of this sgRNA is 5'CCN 3' The design of this sgRNA is according to Matthew H Larson. 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.

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).

To use this part, this part will have to be cloned into a vector consisting of a desired promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]