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).

Results

Gene silencing
We have successfully silenced the McyB gene in Microcystis Aeruginosa UTEX 2388 using this construct. We cloned BBa_K3219000 into a shuttle vector with CaMV35S RNA promoter and ribosome binding site. After 3 weeks after transformation of the shuttle vector with part BBa_K3219000, the Microcystin concentration was lowered compared to the control set-ups.

Figure 1: Microcystin detection kit sample

Figure 2: Our results:
1st test (from left): Culture of Microcystis 3 weeks after transformation
2nd test: Water
3rd test: Culture of unsuccessful Microcystis transformation after 3 weeks
4th test: Positive control of Microcystis culture that has not been transformed

These results indicate that our successful transformation of Microcytsis has a Microcystin concentration lower than 0.002mg/L, while our positive control set-ups have a higher concentration, showing positive results. The experimental set-up and control set-ups were treated under the same conditions to ensure a fair test. The results show that the dCas9 was expressed and was able to silence the McyB gene.

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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