Team:Stanford-Brown/Projects/CRISPR

Introduction

CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins.

The most commonly studied system is the self-contained Cas9 protein from Streptococcus pyogenes, which has drawn increasing attention for its use in genome editing. We sought to BioBrick the Cas9 protein and variants for a different purpose — programmable transcriptional regulation. By conjugating a plasmid containing Cas9 and a targeting CRISPR, we aimed to regulate the genes of an entire population for therapeutic applications.

Lab Notebook

Biobricks

[http://parts.igem.org/Part:BBa_K1218011/ BBa_K1218011 (pCas9)] This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.

[http://parts.igem.org/Part:BBa_K1218019/ BBa_K1218019 (CASCADE Complex)] This polycistronic sequence contains the CasABCDE genes that form the CASCADE complex. Functions in the innate CRISPR-Cas immune system of Escherichia coli.

[http://parts.igem.org/Part:BBa_K1218014/ BBa_K1218014 (dCas9-ω Activator)] This part codes for the tracrRNA, a mutated Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9. However, the Cas9 protein is mutated or "dead" and is fused to a RNAP omega ("w") subunit. Binding of the Cas9-RNAP-subunit fusion activates transcription of the targeted gene.

Data

We submitted bricks for Cas9, a minimal CRISPR array, and transcriptional activator variant of Cas9, dCas9-ω, the first complete, BioBrick compatible CRISPR-Cas system. Our characterization of the bricks is pending while we construct a suitable fluorescent targeting constructs.

Applications