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 to join and we're excited to hear what you guys think of it! Sign up at: info.abmgood.com/crispr-crash-course
Hi Keira, glad you enjoyed the video! Good luck on the project! If you want to know more about CRISPR, we have a knowledge base which you can find here: goo.gl/rSZuAZ
Additionally the repression/activation protiens can be engineered to bind to an extention of the guide RNA's so that multiple reactions can take place simultainously. The dCas9 shown here would only be good for one reaction at a time, where all genes targets are activated or repressed but cant do both for different sets of gene targets. RNA binding components are able to do that though and is also an option.
As far as I know, spacer sequence (in sgRNA) goes and binds to the DNA strand, opposite to the DNA strand that contains PAM sequence. in the video it shows the other way around. Could you please correct the video or me (If i'm mistaken).
Hi Chamara, you're right that the sgRNA binds to the opposite DNA strand that contains the PAM sequence. This is actually what we are showing in our video - but indeed, we could have been more clear by associating the sgRNA closer with the opposite DNA strand. Thanks for your comment - we'll take note of this for future videos!
For example, I discovered a bacterium producing an anti-quorum sensing compound. How to use CRISPR technology to explore the gene involved? I hope for an immediate response for this question. Thank you very much.
Hello Alvin! The CRISPR system can be used to perform gene editing experiments such as gene knockouts in which a gene is made inoperative. Typically, scientists would knock out a gene to confirm/observe/learn more about the gene's function by comparing the knockout organism to the wild-type/normal organism. In your case, you could use the CRISPR system to perform a screening experiment, in which you generate a pool of sgRNAs targeting knockout of different genes and deliver them into your bacterium. You can then screen your pool of mutant cells for the phenotype of interest - in this case perhaps the gene behind the anti-quorum sensing compound. CRISPR screening experiments like this can be used to identify previously unknown genes that contribute to a phenotype.
Thanks for this simple overview. In the video and other videos, people call the dCas9 a reversible system, but they never explain how one removes/prevents the crispr cas9 system from working to reverse the effects? Either a simple explanation or some terms or method names I could look into?
Hi Andries, thanks for watching and leaving a comment! The dCas9 null mutant is being described as "reversible" in this context because it has the potential for its effect to be reversed. In comparison, when the fully operational Cas9 nuclease edits the genome, this change is permanent, even if the Cas9 protein stops being expressed. dCas9's "reversibility" can be taken advantage of in a few ways. The simplest way to achieve reversible dCas9 effects is by only transiently expressing the sgRNA. Once the sgRNA has become diluted, dCas9 will be unable to bind to its target and it's function will cease. One method to control dCas9 is by expressing it under an inducible promoter (see www.nature.com/articles/srep39076 for an example). The effect of dCas9 can be stopped simply by washing the cells to remove the inducer. Once expression of dCas9 is stopped, the protein becomes diluted and its effects will lessen until it is completely gone. Other techniques inducibly recruit effector domains to dCas9 (here are two examples: (www.nature.com/articles/s41467-017-00644-y, www.ncbi.nlm.nih.gov/pmc/articles/PMC4412021/). These may be reversed by adding a competitive inhibitor of the effector complex or by removing the inducing substance. Here is a review of the types of inducible CRISPR editing that are available, some of which are reversible: www.tandfonline.com/doi/abs/10.1080/07388551.2017.1378999?scroll=top&needAccess=true&journalCode=ibty20 You can also check out our knowledge base for more info about the dCas9 system and its possible uses for gene regulation: goo.gl/rSZuAZ
Hi, thanks for leaving a comment! You can clone in the Cas9 gene into your expression vector of choice using standard cloning techniques (at abm, we offer a wide range of Cas9 expression vectors: aav, lenti, adeno, etc.). Such a system can then be transfected into the cell for gene editing along with an expression vector carrying your guide RNA.
why can't they use crisper cast 9 to cure diseases help people walk and improve life and keep people dying and they HIV and stuff like that and muscular dystrophy and forget about designer babies and all that stuff that's them it could help us out so much please hurry as fast as you can keep me updated thank you
Hi Larry, while CRISPR shows a lot of potential for curing diseases in humans, there are still many obstacles, such as eliminating off-target effects, and addressing legal and ethical concerns. This is a helpful article from the Genetic Literacy Project that explores some of these obstacles: geneticliteracyproject.org/2018/04/02/whats-stopping-us-from-using-crispr-to-gene-edit-humans-to-fight-disease/ So far, there has only been a handful of approved CRISPR clinical trials, such as this one that is aimed to treat β-thalassemia: www.the-scientist.com/news-opinion/us-companies-launch-crispr-clinical-trial-64746. Here's another article from Nature that covers the upcoming first in vivo trial of CRISPR medicine in humans: www.nature.com/articles/d41587-018-00003-2
Your questions about CRISPR are welcome! Post them here and learn more about how you can epigenetically activate genes using this special Cas9 double mutant at our knowledge base: goo.gl/rSZuAZ
