Team:Stanford-Brown/Projects/CRISPR

Introduction

What if we could immunize bacteria from becoming antibiotic resistant? What if pathogens exchanged a plasmid that prevents them from causing disease? What if we could regulate entire families of genes simultaneously using short stretches of DNA?

With CRISPR-Cas, we can.

CRISPR-Cas bacterial immune systems are capable of remembering and targeting invasive DNA by storing short DNA sequences, called spacers, in genomic arrays called CRISPRs. RNA transcripts of the spacers are then incorporated into CRISPR-associated (Cas) proteins, which surveill the cell and degrade DNA complementary to the spacer.

The most-studied CRISPR-Cas system is the Cas9 protein from Streptococcus pyogenes, which has drawn much attention for its utility in genome editing. We sought to BioBrick the Cas9 protein and variants for a different purpose — programmable transcriptional regulation. By using a "deactivated" Cas9 mutant dCas9 that retains DNA-binding but not degrading activity, we sought to brick a transcriptional repressor that can be directed to arbitrary regions of the genome (Bikard et al., 2013). We also sought to brick the dCas9-ω transcriptional activator, which contains the omega RNA polymerase subunit fused to the carboxy terminus of dCas9 (Bikard et al., 2013).

To extend on previous work using Cas9 as a transcriptional regulator, we sought to conjugate it between cells to regulate specific target genes in an entire population. We envision the system as means to combat antibiotic resistance by degrading resistance plasmids or even rendering the pathogen harmless.

Protocols

A complete list of our protocols can be found here.

Lab Notebook

We documented our experimental work in an online notebook.

Data

We submitted bricks for Cas9, a minimal CRISPR array, and transcriptional activator variant of Cas9, dCas9-ω. To our knowledge, the bricks constitute the first complete, BioBrick compatible CRISPR-Cas system submitted to the registry. Our characterization of the bricks is pending while we construct fluorescent testing constructs to demonstrate the cleaveage activity of Cas9 and regulatory activity of dCas9-ω when introduced via conjugation.

We also demonstrated successful conjugation of the RP4 plasmid. Electrocompetent cells were transformed with RP4, a plasmid coding for proteins needed for conjugation, and grown in liquid culture containing ampicillin. The ampicillin-resistant RP4 cells were mated with cells containing chloramphenicol-resistant RFP, and the resulting strain was plated on a double antibiotic amp+chlor plate. The two parent strains were also streaked to confirm that each did not possess the needed resistance genes prior to conjugation.

After conjugation was confirmed, we performed a midiprep of the RP4 cells to obtain the RP4 plasmid. Later, this RP4 plasmid will be transformed into our donor cells to facilitate conjugation of the CRISPR-Cas system.

Applications

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.

Acknowledgements

We would like to thank:

• Dr. Lynn Rothschild, Dr. Gary Wessel, and Dr. Joe Shih - our primary advisors
• Dr. David Bikard1 - for providing Cas9 plasmids and clarifying questions about promoter choice
• Dr. Leonard A. Mermel - whose paper provided one of the primary inspirations for this project
• Dr. Kosuke Fujisima - for mentoring us in the lab
• Jane Berry at EMD Millipore
• Coli Genomic Stock Center [CGSC]

References

• Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research.
• Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., ... & Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607.
• Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology, 31(3), 233-239.
• Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821.
• Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., ... & Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science, 339(6121), 823-826.
• Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183.
• Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell.