Gene Silencing Methods: CRISPR vs TALENs vs. RNAi

Although the CRISPR system originated in bacteria, it is more commonly used to edit eukaryotic genomes rather than bacterial genomes. This is because most bacteria are unable to effectively repair double-stranded DNA breaks created by Cas9. Scientists have therefore developed a method that combines both recombineering technology and CRISPR Cas9 to allow us to be able to edit bacterial genomes!

To demonstrate how this can be done, we use the CRISPR Cas9 system to knockout the chloramphenicol resistance casette (CAT) gene in E. coli (previously introduced into the genome at the yeeR locus). This process involved:

1. designing and delivering sgRNAs against the target site,
2. designing and delivering a repair template,
3. delivering phage-derived (lambda red) recombinases alongside Cas9 to enhance homologous recombination in bacteria.

E. coli transformants were screened for sensitivity to chloramphenicol. The knockout was then verified using a restriction enzyme digest method and confirmed via Sanger sequencing.

We hope this video can help you in designing your own bacterial knockout experiment! Please feel free to leave your questions in the comments and we'll be happy to answer them!

Read other case studies using CRISPR Cas9:

Learn more about how abm's suite of CRISPR Cas9 tools can assist you in your gene editing projects:

Read our complete Bacterial CRISPR Knockout Case Study here:

For a list of theoretical and practical CRISPR resources:

*NEW -This E. coli CRISPR Knockout experiment is now available as a teaching kit! Contact us for more information on how you can do CRISPR in the classroom.

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[cacevedo]

Excellent video. Many thanks.
Do you have a tutorial on how to make cells stable for Cas9 expression? I guess this is the first step before doing the first viral transduction?

[simranrachel]

Thank you very much, best palylist for know crispr ....💖💖💖💖💖its going to help a lot for my investigatory project at school

[Dabby]

0:52 Currently I'm reading a lot about double strand break repair mechanisms in bacteria and now I'm wondering if this information in the video at this timestamp is actually valid :/

[farahbourek]

I want to ask about methods of extraction of crispr cas9

[thefuturedoctor]

Great video! What approach would one take to make a scientific breakthrough using the CRISPR tool?

[frpcsge]

Hi I'm new to this field. I left school due to health problems. I decided to self teach and research myself. I'm kind of lost, what are my requirements to confidently follow the procedure?

[Family_FSRA]

I have a query. How do i insert the repair template? Should we have to clone the repair template into any plasmid vector or the amplified PCR product will do the job?

[idowubello-osagie]

Hi there. Do you have any video on gene editing in fungi e.g Aspergillus niger?

[andyherreragarcia]

can this technique be applied to Ensifer meliloti? we are trying to make knockouts of new genes

[martinjeffers]

Can this technique be applied to Klebsiella oxytoca?

[usingh]

Thanks for the video.

Was curious to know whether you would be able to design an appoach for gene editing e. coli (or yeast) so that it can be used to produce collagen (from plant based products) - for vegan cosmetics?

[uberawsome]

Thanks a lot. I just signed up for the course and was wondering if the actually doing it was nessary. Beacuse as a high school freshman I do not have access to all the nessary stuff.

Also will it cover adeno associated viruses.

[ocm]

I'm looking to use CRISPR-cas9 in conjunction with HDR to perform a knock-out/knock-in mutation in a Pectobacterium strain. I do have the lambda red recombineering system as a back up in case this fails, but hoping that CRISPR will work for me here. Any advice on implementing this for a bacterial system?

[debasishbiswal]

Thanks a lot for this very informative video. I would like to ask you if you can help me doing an experiment on a gene knockout experiment in Kocuria flava (a gram positive bacteria). Thanks in advance and Your reply will be highly appreciated