Gene Silencing Methods: CRISPR vs TALENs vs. RNAi
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- Опубликовано: 3 окт 2024
- 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:
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Hey everyone! If you're interested to learn more about CRISPR, we'd love for you to check out our new 4-week CRISPR Crash Course. It's completely free and you'll be ready to perform a successful CRISPR knockout experiment by the end of it. Sign up at: info.abmgood.com/crispr-crash-course
Could this method also be used for positive selection??
Hi Kat, absolutely! In that case, you would provide a repair template with a selection marker flanked by homology arms. I recommend you check out our Bacterial CRISPR Knock-In Case Study (www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Case_Studies.php#BactKI). In the case study, we knocked-in a mCherry gene which allowed for visual screening, but you could do a similar experiment with an antibiotic resistance gene, instead.
Subtitle to spanish, please?
Thank you very much , best palylist for know crispr ....💖💖💖💖💖its going to help a lot for my investigatory project at school
Hello Simran,
We are glad to hear that you found our video helpful! Stay tuned for our upcoming videos as well! :)
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?
The method outlined in this video targets E.coli. In this case, λ red (a phage-derived recombinases) is used to improve the recombination rate and it is included in the pCas plasmid.
If you are working with mammalian cells, one common way is to infect cells with lentiviruses as lentiviruses are capable of integration into the host cell's genome. For further information, feel free to contact us at technical@abmgood.com.
Thank you! :)
Great video! What approach would one take to make a scientific breakthrough using the CRISPR tool?
This Crispr-cas9 protocol can cure for every kind of rare, genetic diseases. This is holy and human supportive job by these scientists.
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?
Hello,
We can definitely help you! Please contact us at technical@abmgood.com to discuss in detail. Thanks! :)
I want to ask about methods of extraction of crispr cas9
Hi there. Do you have any video on gene editing in fungi e.g Aspergillus niger?
Hello there,
Unfortunately, we didn't have a chance to create a video on gene editing in fungi yet but it sounds an interesting topic to cover! We will definitely let our content production team know :)
can this technique be applied to Ensifer meliloti? we are trying to make knockouts of new genes
Hi Andy, the particular system that is described in the video is optimized for usage in E. coli and will likely not work with this organism. It is primarily based on the recombineering system lambda red derived from lambda phage, and literature shows that this system has not been developed or successfully used in Ensifer (Rhizobium) species. A system similiar to this could probably work for many different organisms, with the system consisting of native or non-native recombinase + CRISPR. The challenge would be to design compatible plasmids and other genetic elements tailored for your organism. Hope this helps.
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?
Yes, we offer custom gene editing services. Please contact us at technical@abmgood.com for more details if you are interested!
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 :/
Hi Dabby3,
Thanks for watching our video and leaving your comment!
Most bacteria cannot easily or efficiently repair double-stranded breaks in their gDNA - which is the case for E. coli. For example, E. coli prefers to repair double-stranded DNA breaks via homologous recombination and its native system is not very efficient, this is why scientists implemented the phage Lambda red system into this organism in the first place (pre-CRISPR era). When double-stranded breaks are present in the gDNA, the cell's normal metabolic functions stall until the break is repaired, which includes growth and division. The idea is that Cas9 overwhelms the native repair system as it is constantly targeted to the site and cleaving the DNA. This of course, is not a 100% foolproof counter-selection method, as bacteria are adept to changing environments. After transformation we typically detect a very small portion of what are called 'escapers' which are E. coli cells that have managed to mutate their gDNA in a way to avoid damaging Cas9 cleavage.
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?
Hi Feroz, for Bacterial CRISPR, the options for HDR repair template are quite flexible. The repair template can be provided as a single stranded oligonucleotide, double stranded oligonucleotide, a double stranded DNA fragment (like a PCR amplicon), or a plasmid.
Plz Crispr-cas9 protocol for polycystic kidney disease thanks
Can this technique be applied to Klebsiella oxytoca?
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.
Hi, thanks for watching and leaving your comment! You don't need to do anything hands-on to complete the course. The course is meant to be a practical guide for how you could carry out a CRISPR gene editing experiment. Currently, we only have the lentivirus guide but in the future, we may make one for AAV!
@@abmgood thanks also can you use aav and the like to modify organs or are bacteria the only thing.
Yes, researchers can use AAV to deliver CRISPR components for in vivo genome editing in animal organs/tissues!
Thank you so much for letting me know i want to try to modify the organ that makes insulin. Would that be possible
AAVs come as different serotypes that can target different tissues, so it is possible to target the pancreas which produces insulin. There is still ongoing research on which serotype(s) is the best for targeting certain tissue types, but there is a lot of literature out there that you can explore!
We also have a in-depth knowledge base on the AAV system if you want to learn more: www.abmgood.com/marketing/knowledge_base/Adeno_Associated_Virus_Introduction.php
Hi there, thanks for the informative video. I'm under the impression that the CRISPR-cas9 system can be used to some extent to modify bacterial DNA. Here's a paper I've glanced over that seems to back this up: www.ncbi.nlm.nih.gov/pubmed/23360965
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?
Yes, CRISPR-Cas9 system can be used to modify bacterial DNA. Check out our bacterial CRISPR services here: www.abmgood.com/CRISPR-Bacterial-Genome-Editing.html, our services currently cover E. coli. species only. For Pectobacterium strains specifically, you may find this paper helpful: pubmed.ncbi.nlm.nih.gov/24256239/
Is there Crispr-cas9 protocol for polycystic kidney disease to knock out PKD1 pkd2 genes thanks
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