Contribution

As part of our project, we produced two types of contributions to disseminate knowledge to future iGEM teams. The first one is a registry for Streptomyces strains and a list of protocols that we call the 'STREPTObook'. We hope that it will allow future teams to start experiments on these fancy chassis! The second contribution concerns the elaboration of a tutorial for the design of CRISPR tools dedicated to gene silencing, an innovative and rapid application to modulate gene expression. Below we give you our iGEMers secrets about this CRISPR design!

Why a CRISPRi tuto for gene silencing?

As our goal was to build a chassis producing antibiotics from captured CO₂, we had to consider both, the efficiency of carbon fixation, as well as the efficiency of production antibiotics. To do it, we thought of not only adding genes to Streptomyces' genome, but also silencing existing genes that hinder our desired outcomes (better implantation of CO₂ Calvin cycle, unsilencing of antibiotic biosynthetic gene clusters). That is why, we decided to use the CRISPR dCas9 system, which allows for a very precise silencing of regions of interest, thanks to its unique feature, which is the guide RNA.

Guide RNA, which along with the dead Cas9 enzyme, make up the CRISPR-dCas9 system. It is complementary to the target sequence, thus allowing the complex to bind to a specific location, which can then inhibit its transcription. Thus, the design of the guide RNA sequences is crucial for the functionality of the silencing system.

To design our sequences, we used multiple useful resources, which were recommended to us by our coaches. However, not all iGEM teams have the luxury of being guided by people who have already used this system, which may be a major setback for them, since the CRISPR Cas9 is quickly becoming a major tool in synthetic biology research. That is why, at the team GO Paris-Saclay, we have decided to create a comprehensive resource containing helpful links, videos and written documents, to guide future iGEM teams in the construction of their own guide RNA sequences for the CRISPR dCas9 system.

We hope that this contribution allows new iGEM competitors to achieve their projects’ objectives and enrich the competition’s database.

// CRISPR dCas9 tutorial //

Published on 19.08.22

Contents:

Introduction

CRISPR-Cas9 is a molecular scissors complex composed of an RNA strand and a nuclease that has been widely discussed in scientific research in recent years. In 2020, the development of a gene-editing system using CRISPR-Cas9 has earned Emmanuelle Charpentier and Jennifer Doudna a Nobel Prize in Chemistry. CRISPR-Cas9 has found a wide variety of applications in synthetic biology beyond its original purpose, one of which - gene silencing, we will discuss in greater detail in this tutorial.

Origin and Structure

Originally, CRISPR-Cas9 is a system of defense found in bacteria and archaea that is targeted against phages and other foreign DNA sequences such as plasmids. The system functions as a highly specific pair of molecular scissors, the guide RNA sequence fixes to the phage DNA and the Cas9 protein cleaves it in two places.

The origin of the guide RNA sequences are the phages themselves. Some bacterial species have adapted to integrate the DNA of phages that have infected them; that DNA provides a template for future guide RNAs to build “immunity” against this phage.

But why don’t the scissors cut the same sequences in bacteria? That is all thanks to PAM. PAM or a protospacer adjacent motif is a small sequence that is present in the phage genome, just after the targeted sequence. It is recognized by Cas9 and the enzyme will not cut the DNA, unless there is this three nucleotide long sequence present, and the bacterial genome simply does not contain it.

Gene silencing with CRISPR-Cas9

Beyond its function as a pair of molecular scissors, CRISPR-Cas9 can also be used as a gene silencer. To achieve it, scientists have mutated the Cas9 nuclease to prevent it from cutting the targeted DNA. This new version has been called a dead Cas9 or simply dCas9. It functions in the same way as a regular Cas9, fixing to the targeted sequence but not damaging it. Therefore, if the targeted sequence is chosen correctly, the CRISPR-dCas9 can fix to a specific part of a gene and silence its expression without resorting to knocking-out the gene.

In this tutorial, we will try to show you how to design a guide RNA for the CRISPR-dCas9 system, to silence the activity of a desired gene.

The targeted gene and the chassis

Once you have chosen a gene and the organism you want to silence it in, you have to download the genome of the chassis on the NCBI website. You need to type in the name of the organism into the search bar and download the genomic sequence in the FASTA and GenBank formats. In order to do that you need to scroll down on the site and find “Reference Genomes'', then click on the “RefSeq”. To download the sequence click on the “send to” function in the top right corner then Complete Record>File>GenBank (full)>Create File and Coding Sequences>FASTA nucleotide>Create File.

After that, you need to find the location of the gene you want to silence.

AntiSMASH

If the gene codes a secondary metabolite (antibiotic etc.) a great resource for it is the AntiSMASH website (Blin et al., Nucleic Acids Research, 2021; doi: 10.1093/nar/gkab335). You upload the FASTA or GenBank file and click “Submit”. You choose the region of choice, then the specific gene, preferably a core biosynthetic gene and note its location. In order to download the sequence of this gene, you need to go back to the same NCBI website you downloaded the chassis genome from. To download a specific region of the genome, you need to change the “selected region” in the “Change region shown” box on the right. Indicate the beginning and the end of the gene, then proceed using the send to function, as explained previously.

NCBI

If your gene of interest has no link with specialized metabolism, remain on the NCBI website and scroll down to the same Reference Genome table. Find the column “Protein” and click on the number below it. You should find a table with all the genes found in your chassis' genome. Type in the search bar the name of the protein or locus of the gene to find it and note the location of the gene. Then, click on the number in the “Protein Product” column and download the FASTA sequence of the gene using the “Send to” function, as explained previously. All this information will be needed in future steps.

How to find the guide RNAs?

There are multiple websites that help to find the appropriate guide RNA for your CRISPR-dCas9 system. We will present two of them and we recommend using both and comparing the results to find the best guide RNA.

The CRISPy website (Blin et al., Synthetic and Systems Biotechnology, 2016; doi:10.1016/j.synbio.2016.01.003) is a great resource to find the appropriate guide RNAs, not only for silencing genes, but for any other use, it shows the number of off-targets with their degree of similarity, which is important to consider while choosing the best sequence.

To use the website, you need to upload the genome sequence in the GenBank format and then indicate the region of interest (location of the gene). The website shows the proposed guide RNAs and ranks them according to the number of off-targets. For silencing a gene, the guide RNA needs to be preferably complimentary to the coding sequence, thus you need to be looking for a sequence with a “-” in the strand column.

The Pasteur website (Calco-Villamanan et al., Nucleic Acids Research , 2020; doi: 10.1093/nar/gkaa294) is also a great tool for designing a guide RNA. It is especially useful because it attributes sequences scores calculated according to the model mentioned on the site. In order to use the site, click on the “Guide RNA design” button. Then, paste the sequence of your target gene that you previously downloaded in the FASTA format, just open the file and copy paste the sequence. Then, upload the genomic sequence either in the FASTA or GenBank format, preferably FASTA, it uploads faster. Click “Run” and wait. The sequences are ranked according to their silencing abilities; the number of off-targets is shown on the right.

To find the best results, we recommend comparing both of the sites. The Pasteur website is better at showing the silencing abilities of the guide RNAs, while CRISPy is more reliable when it comes to off-targets. Now you are ready to order your first CRISPR-dCas9! We hope you found this tutorial helpful!

If you used this tutorial, please contact us!

or in case of any questions regarding the tutorial, contact me via bartosz.fraczek@universite-paris-saclay.fr