Part:BBa_K3219001

sgRNA for mcyB Microcystis Aeruginosa UTEX 2388

Introduction

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).

Usage

To use this part, this part will have to be cloned into a vector consisting of a desired promoter. Also, the sgRNA should be put together with a dCas9 enzyme in order to achieve the desired repression of McyB gene.

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]

↑ Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jennifer A. Doudna, Jonathan S. Weissman, Adam P. Arkin, Wendell A. Lim. “Repurposing CRISPR as an RNA-Guided Platform for SequenceSpecific Control of Gene Expression.” Cell (2013): 1173–1183. Journal.
↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.
↑ Matthew H Larson, Luke A Gilbert, Xiaowo Wang, Wendell A Lim, Jonathan S Weissman, Lei S Qi. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. California: Nature America, Inc. , 2013 .

[1] Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jennifer A. Doudna, Jonathan S. Weissman, Adam P. Arkin, Wendell A. Lim. “Repurposing CRISPR as an RNA-Guided Platform for SequenceSpecific Control of Gene Expression.” Cell (2013): 1173–1183. Journal.

[2] Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.

[3] Matthew H Larson, Luke A Gilbert, Xiaowo Wang, Wendell A Lim, Jonathan S Weissman, Lei S Qi. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. California: Nature America, Inc. , 2013 .

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