BE FPOP(Su24):Identifying CRISPRi targets
Contents
Introduction
For your first exercise you will study the pathway used by E. coli to produce ethanol and hypothesize which genes can be targeted to increase yield. Before you hypothesize on which genes might make good targets, it is important to learn more about how the CRISPRi system works.
The CRISPRi system is programmed using sgRNAs, or single-guide RNAs, that are complementary to the DNA sequence of a target site within the genome. In our experiment, the sgRNAs are complementary to the non-template strand of the target genes. When the sgRNAs complex with the dCas9 protein, the sgRNA directs the dCas9 protein to bind at the target gene. This binding prevents RNA polymerase from transcribing the target gene which leads to a decrease in the protein that is encoded by that gene. This method of action is referred to as the collision model.
Protocols
Research CRISPRi system
Review and discuss the Introduction from the following journal article with your laboratory partners:
Lei et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell. (2013) 152:1173-1183.
In this article, the researchers describe a modified CRISPR system, referred to as CRISPR interference (CRISPRi), that inhibits transcription of genes that are targeted using single guide RNA (sgRNA) molecules. The goal of this exercise is to understand how the sgRNA molecules are used to target specific genes. In the CRISPRi system, sgRNA forms a complex with dCas9 and this complex binds to the sequence in the genome that is complementary to the sgRNA molecule. Thus, the sgRNA targets a specific gene sequence in the host genome. When the sgRNA / dCas9 complex binds, transcription of the gene is inhibited.
Transcription can be blocked by targeting sgRNA molecules to the promoter or coding region of a specific gene. The promoter is the region upstream of the start codon and contains the RNAP binding site, or the -10 and -35. When the sgRNA / dCas9 complex binds to the promoter, transcription initiation is inhibited because RNAP is unable to bind to the promoter. The coding region refers to the gene sequence. When the sgRNA is targeted to the coding region, transcription elongation is stalled because RNAP is unable to traverse beyond the location of the sgRNA / dCas9 complex.
In your laboratory notebook, complete the following with your partners:
- In the first paragraph of the Introduction the authors note that other methods are available for genetic manipulations.
- What is a limitation of using RNA interference?
- What is a limitation of using TALE proteins?
- In the second paragraph the native CRISPR system is described.
- What two RNA molecules are required for native CRISPR systems?
- How were the RNA molecules modified for use in engineered CRISPR systems?
- In the third paragraph the engineered CRISPRi system is described.
- How was the Cas9 protein modified for use in the engineered CRISPRi system?
- The components for the CRISPRi system are depicted in Figure 1.
- What three domains are present in the engineered (chimera) sgRNA?
- What is the outcome for each of the engineered CRISPR systems shown? (hint: what happens to the DNA?)
Review E. coli anaerobic fermentative metabolism
Across many fields of science, E. coli is a valuable tool in research related to genetic systems, metabolic pathways, and physiological responses. It is often called the 'workhorse' of bench science. There are many reasons for this role including: 1) E. coli cultures are easy to grow in the laboratory, 2) E. coli is a genetically tractable system, 3) E. coli metabolic pathways are defined and, 4) E. coli is a well-studied organism in the literature.
We will take advantage of the genetic tools available for engineering the genetics of E. coli to increase ethanol yield. In E. coli, ethanol is produced via the mixed-acid pathway which is used by cells under fermentative conditions to maintain redox balance. With your laboratory partner, review the metabolic pathway map below and identify at which step ethanol is produced.
In your laboratory notebook, complete the following:
- Which gene(s) is / are responsible for ethanol production? Provide a brief description of the reaction (i.e. what is the substrate? what are the biproducts that are generated?).
- Which gene(s) might you target using the CRISPRi system to increase the availability of substrate for ethanol production?
- Which gene(s) might you target using the CRISPRi system to decrease the amount of substrate used to generate products in steps downstream of ethanol production?
- From the list of possible gene targets, which do you think is the most promising candidate for increasing ethanol yield? Why?
