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
Check out our newest video about CRISPR/cas9 and the methods and tools available to put this amazing procedure to use! - th-cam.com/video/INC_kdr7I34/w-d-xo.html If you haven't seen our previous CRISPR/cas9 introduction video be sure to also check it out! - th-cam.com/video/1aJxXWkE3Ek/w-d-xo.html
Thank you ! I think it's a "brief" for experts :) If I hadn't learned about this from other videos before, this would have been some high-level information. But when I saw this detailed series, you cannot imagine how I got excited.
Have you seen our video on the methods and tools available for using the CRISPR Cas9 system in your research? - th-cam.com/video/INC_kdr7I34/w-d-xo.html
Great video! If I wanted to knock out a gene in C. diff by using bacteriophage deployment of the cas9 protein, complementary gene sequence, and the guide rna, how would I do so?. A few more questions are: 1. Which of the 3 viral delivery methods would be bacteriophage? 2. How would I go about designing the dna strand that I want to insert? 3. How would I get the edited bacteriophage to grow clones? 4. How can I assure that the bacteriophage will target the C. diff? 5. How can I tell if c. diff uses the Nhej method or the hdr method of dna repair? 6. What can I use to find the base sequence of a particular gene in the c. diff genome? Sorry for all of the questions!! I hope that you all can help 😁
Hello abm, I've been enjoying your videos on CRISPR and, if you have the time, I would be very grateful if you could answer a few questions I have on the terminology used! 1. What is a primary cell? 2. Why can adenoviruses affect primary cells and not lentiviruses? 3. Other than dividing/non-dividing/primary cells, what other types of cells are there? 4. What does 'differential tropism' mean? 5. How does differential tropism make AAV a candidate for regenerative medicine? Thank you for your patience.
Hi Natsukashii, Thanks for watching our video! To answer your questions: 1. Primary cells are cells that are collected directly from living tissue and then grown in vitro. Scientists may prefer using primary cells for their research because they their characteristics are close to how the cell actually behaves in its native environment (as opposed to immortalized cells that have undergone so many population doublings that they may have altered characteristics). 2. Both adenoviruses and lentiviruses have the ability to infect many cell types, both dividing and non-dividing. The adenovirus, however, has a higher transduction rate towards primary cells. 3. Primary cells can fall under dividing or non-dividing cells depending on what cell you are looking at. For instance, neurons are considered non-dividing cells as they are fully differentiated. On the other hand, glial cells and other supporting cells of the brain do have the ability to replicate and are thus considered "dividing cells". Aside from Primary cells, other cell types are immortalized cells, stable cells, and stem cells. 4. Differential tropism likely refers to the idea that certain viruses can infect certain types of cells/tissues. For example, AAV has many serotypes (variations) that enable it to infect a broad range of tissues whereas other viruses like the rabies virus primarily infects neuronal tissue. 5. Because different variations of AAV have a capacity to infect different tissue types, gene therapies can be delivered to very specific locations or to most all tissue types, depending on the need. For example, AAV Serotype 2 has been used in cancer treatments to kill a diverse group of cancer cells without showing any effect on healthy cells. There's more information about AAV on our knowledge base if you'd like to learn more: goo.gl/F7lvrL
Vitalie Samoil Glad you found the video helpful! If you're interested in learning more, we have an additional video in the CRISPR series on gRNA design (th-cam.com/video/dXPDefej0Ps/w-d-xo.html).
Hello. I am working with CRISPR to delete a functional gene from Salmonella. Once the gRNA has been designed, is it possible to include them along with the Cas9 enzyme in a plasmid before being transfected into the Salmonella ?
Hi Najwa! Thanks for watching our video and thanks for leaving a comment. Yes, it is possible to deliver both the sgRNA as well as the Cas9 in the same plasmid to be transfected into your target organism/cell. At abm, we offer "All-in-One" plasmids that can do the trick! May I suggest you take a look at our Genome-wide sgRNA Library to see if we have something of use for you? goo.gl/RpdVZb
These non-viral methods talk about the insertion of RNA into the in-vitro cell, or can those chemical transfections be done inside a living animal? (If so, on what scale? A single tissue, a single organ, or a full organism?) And are these methods (both viral and non-viral) good for the RNA insertion and Cas9 protein as well? Or is the Cas9 inserted in a form of RNA and the cell produces the Cas9 protein from the inserted RNA?
Hi there, thanks for watching our video! Yes, CRISPR Cas9 can be used to modify the genome of a living animal -- this can be done on a tissue-specific level (using viral gene delivery methods like AAV) or to an entire organism (creating transgenic organisms by injecting CRISPR/Cas9 machinery into the embryo). The methods described in the video are commonly used to delivery either the sgRNA, the Cas9 protein, or both into the target cell -- it depends on what you'd like to achieve. For instance, if you'd like to have the cell stably express Cas9, you could use a lentiviral delivery of the Cas9 gene to stably integrate the gene into your cell's genome, allowing it to constitutively express Cas9. Then, all you'd need to do to begin genome editing is to provide the sgRNA targeted to your gene of interest (you could do this by delivery of the sgRNA plasmid via electroporation for transient genome editing). If you'd like more information about how to plan your genome editing experiment, visit our knowledge base: goo.gl/df55ap
If I understand correctly your response, you are saying that you can deliver the Cas9 protein in the lentivirus? (I'm confused by two opposed meaning sentences in your explanation above.) Isn't that too large for the virus or less efficient than using RNA form of the Cas9? I thought the problem with the CRISPR Cas9 is that we have not enough space in the delivery vesicles. Can in-vivo cell create Cas9 out of a delivered version of Cas9?
Hello! I really enjoy your videos about CRISPR/Cas9! I already heard about this method. People say that it's controversal because it would be difficult to tell if there's a GMO or not. In my country, germany, GMO's are under strict conditions. But if you transfer the coding sequences for the Cas9 protein and the gRNA in the target cell, it should be obviously an GMO. So why is it so hard to tell if it's a GMO or not?
Hello Tony! Thank you for watching and leaving a comment! The debate is centered around the difference between what it means to be "genetically modified" vs. "gene-edited". To make a "genetically modified" plant, for example you would add a gene from another organism to that plant (e.g. adding a gene from a bacteria that helps corn be resistant to drought). To make a "genetically edited" plant, you would be editing the plant's existing DNA in order to improve a characteristic of the plant (e.g. deleting a few nucleotides in the mushroom's DNA so that it doesn't turn brown as quickly). Current regulations only include the definition "genetically edited" and therefore so far, in the USA, at least, some CRISPR gene-edited plants have been given the green light to be grown and maybe even sold without too many regulations in the way.
Also, there are many ways to do gene editing in an organism without needing to incorporate the CRISPR Cas9 gene into the cell itself! You can transiently deliver Cas9 and gRNA to temporarily edit the cell before the cell gets rid of it naturally. See our knowledge base for more details: goo.gl/df55ap
Hi Tt Cooper, thanks for watching and commenting! Yes, it is possible to package a 5.1 KB construct. With AAV, the success of packaging large constructs >4.4 kb is sequence specific and can often result in a significantly lesser yield. We mention 3.4 KB as the packaging capacity as, in our experience this does not compromise yield.
The cas9 from S.aureus and S.pyrogenes show infectious diseases in humans,so how can we redesign cas9 or which bacterial protein will be suitable other than s pyrogenes and s aureus ..in order to overcome this issue.
Hello! Thanks for the wonderful information. I have to write a research proposal - Crisper based mouse model for cancer theraputics. Can you please guide me.
vasco cluny the most commonly used method relies on using viral vectors to deliver the Cas9 gene and the gRNA sequence to the target cell which is then expressed. However, it is also possible to induce the target cells to take up the Cas9 protein and gRNA sequence (though the efficiency of the transformation will be much lower) - abm offers pre-design viral vector constructs for the Cas9 proteins and a wide range of gRNA sequences: www.abmgood.com/CRISPR-Cas9-sgRNA.html
Hello! Thank you for these videos, they are great! though I have a question.. Is it possible to introduce the complete crispr-cas 9 protein with sgRNA attatched to a target cell instead of the genetic material that will be transcribed and translated into the final protein form? I am attempting to use crispr-cas 9 to knock out a hox gene (CDX 1) in a zebrafish zygote. The ideal situation would be to have the complete Crispr cas 9 protein with bound sgRNA already present in the egg, in an inactive state, and have it activate at fertilization. If I introduce this endonuclease machinery as genetic material to be translated to protein, I suspect that developmental cleavage of the zygote will have begun by the time the crispr protein is translated, leading to only some of the blastomeres will be acted on by Crispr.
Hi Alejandro, thanks for watching and leaving a comment! I believe the CRISPR technology you are referring to are the Cas9-sgRNA ribonucleo-protein complexes (RNPs). I don't believe there is currently a well-documented way of keeping this complex dormant inside the egg until you need to activate it. However this complex begins work quickly after transfection so you can deliver the RNP complexes via microinjection/electroporation to the egg right after fertilization, a common technique that is used to deliver RNPs. We do offer custom CRISPR RNPs, if you are interested -- send us an email to learn more: info [ at ] abmgood.com!
Hello, I may have misunderstood something, but I'm a bit confused. All these methods are described as being used to introduce genes to the cells, but Cas9 is a protein. Do these methods introduce teh cas9, guide RNA, and the gene you are introducing, or only the latter?
It all depends on the delivery method used when performing knock in experiments, where the presence of Cas9, sgRNA and desired repair template (this could be a whole gene or a single nucleotide mutation) are required. Generally speaking, there are two main ways to introduce the components mentioned above for gene editing (insertion of gene or making a mutation in a gene, but not knockout): - Non-viral or non-integrating: This typically is known as the more transient way, where Cas9 is introduced into the cell as a protein, mRNA, or expressed from a plasmid, while the sgRNA is introduced as RNA or expressed from a plasmid. Alternatively, these can also be delivered by a non-integrating virus like AAV or Adenovirus. These are known as non-integrating methods, so once the genomic edit is made in the host cell, the Cas9, sgRNA and repair template shouldn't persist in the cells after several rounds of cell division. - Viral integration: When using integrating viruses such as lentiviruses, the Cas9 and sgRNA sequence are delivered into the cell via viral RNA and usually integrated into the host cell genome along with the rest of the viral backbone sequence (the repair template can be provided separately via transfection or can be included in the viral payload). This will allow persistent expression of Cas9 and sgRNA for a higher success rate for editing, and will also allow for selection of infected clones if a selection marker such as puromycin is included in the viral backbone. Some experiments may not prefer the persistent expression of Cas9 and sgRNA once the editing has taken place, so in these cases, we would recommend going with a non-viral or non-integrating method.
Hi! Thanks for making these videos. I’m new to this technology and I’m trying to understand how it works in real life situations. What I still don’t understand is if I can introduce Cas9 and a gRNA using ONLY a plasmid, even if I’m working with human cells, or if this is the case, can I use ONLY a virus or electroporation for example, to deliver Cas9 and the gRNA?
Thank you for your question and we're glad you're enjoying the videos. If you are working with human cells, you have several options, and we will elaborate on the most common ones for each of the methods you mentioned: 1) DNA Transfection: You can use a DNA transfection reagent to deliver Cas9 and the gRNA to the cell. The Cas9 and gRNA can be supplied on two separate vectors, or can be on the same vector, typically called an All-in-One system. This method highly depends on whether your cell type has a high transfection efficiency. 2) Electroporation: You can also electroporate plasmids into the human cells, but more commonly, scientists use electroporation to deliver recombinantly expressed and purified Cas9 protein with transcribed gRNA. This is typically called a RNP (ribonucleoprotein), though electroporation effectiveness can highly depend on cell type. 3) Virus: This is one of the most effective methods of Cas9 and gRNA delivery, as long as your cell type is susceptible to viral transduction. The virus can be a two virus system (i.e. one virus carrying the Cas9, one virus carrying the gRNA), or it can also be an all-in-one system (one virus carries both the Cas9 and gRNA). If you want to learn more about how to use viruses to deliver Cas9 and gRNA to mammalian cells, you can visit our Case Studies page here which walks you step by step on how a group of researchers used our All-in-One Lentiviruses to achieve mouse cell knockouts: www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Case_Studies.php#Knockout
Hi Nazima, thanks for your comment! Argobacterium delivery of CRISPR/Cas9 into plants has been demonstrated -- there's a paper here you can read on one such experiment: goo.gl/6kCtYj
hi this is Arti. I want to learn this technology for my research work in Algae, so can you suggest me the way how actually this technique works in real time.
Hi Arti, thanks for leaving a comment. We currently don't offer any resources/videos on using CRISPR in plants. We do have "how to" videos demonstrating how to use this technology in mammalian cells (see: How to perform a CRISPR Knockout Experiment - th-cam.com/video/1BSiFfyObTY/w-d-xo.html, or How to perform a CRISPR Knockin Experiment - th-cam.com/video/PlVy1oupFxw/w-d-xo.html), and in bacteria (see: How to perform a Bacterial CRISPR Cas9 Knockout Experiment - th-cam.com/video/7pOR3ssg7dg/w-d-xo.html). I hope this helps.
Hi Tasnuva! If you want to learn more about this topic, I recommend that you check out our CRISPR knowledge base article which covers more in-depth information: old.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Tools_Methods.php
Don't we only need to let the sgrna in ?why are we talking about dna entering ? and finally how can we let the cas9 enzyme in? wish to respond fast.thank you
Most commonly, the sgRNA and Cas9 are delivered into the target cell as DNA sequences on a plasmid. The cell's machinery then performs the transcription to generate the sgRNA, as well as the transcription and translation for the Cas9 protein. The delivery of the genetic DNA sequence can be done using one of 2 ways: 1) DNA Transfection: You can use a DNA transfection reagent to deliver Cas9 and the sgRNA to the cell. The Cas9 and gRNA can be supplied on two separate plasmids, or can be on the same plasmid, typically called an All-in-One system. This method highly depends on whether your cell type has a high transfection efficiency. 2) Virus: This is one of the most effective methods of Cas9 and sgRNA delivery, as long as your cell type is susceptible to viral transduction. The virus can be a two virus system (i.e. one virus carrying the Cas9, one virus carrying the sgRNA), or it can also be an all-in-one system. The viral payload (carrying the Cas9 and sgRNA sequences) then gets integrated into the host cell genome (in the case of lentivirus) or gets transcribed directly from transient DNA-genome viruses such as adenovirus or AAV. A third way that doesn't require the use of DNA is Electroporation. You can also electroporate plasmids into the human cells, but more commonly, scientists use electroporation to deliver recombinantly expressed and purified Cas9 protein with transcribed sgRNA. This is typically called a RNP (ribonucleoprotein), though electroporation effectiveness can highly depend on cell type. If you want to learn more about how to use viruses to deliver Cas9 and sgRNA to mammalian cells, you can visit our Case Studies page here which walks you step by step on how a group of researchers used our All-in-One Lentiviruses to achieve mouse cell knockouts: www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Case_Studies.php#Knockout
@@باسل-ر8ص It is unlikely that cells will reject the plasmid once it is successfully transfected into them. Transfection efficiency and being able to get the DNA into the cells usually plays a larger role in this case, and each cell type responds differently to various transfection reagents and delivery methods (eg. some transfection reagents could be more cytotoxic to some cells, other cells are not as susceptible to chemical methods (transfection reagents) vs. biological methods (viruses)). Once in the cell, the promoters used in the plasmid do play a big role in controlling transcription. You have to make sure the promoters in the plasmid are active in the host cell type you are working with. For example, some promoters are constitutive and active in most cell types, while others are tissue-specific promoters and are only active in certain cell types. Some suppliers (including abm) offer reporter vectors or promoter blast kits where a collection of plasmids with promoters driving a reporter like GFP or RFP can be used to transfect cell lines of interest to find the promoter that is most active in a particular cell line. The best promoter is then selected for use in the plasmid that will express the researcher's gene of interest. As with all experiments, other factors could be involved in transcription and translation once the DNA gets into the cells, so it is always best to verify expression by RT-qPCR or RT-PCR, or by Western Blot when possible.
Can crispr edit genes while you are alive or is it your offspring who only benefit for example if i have a genetic disease and crispr disables that gene will that mean i will be cured as well as my off spring?
The question that you have brought up has many intricacies that needs to be taken into consideration. Most of what we know today about genome editing is from work done on cultured cells. Looking at cells grown in culture, when the genome is altered, that altered genome will be passed down to their offspring. There's a lot more involved when it comes to humans. theoretically, changes made to a somatic cell will only affect the cells involved and therefore the paitent involved, however, if the change is made to gamete, germ cell, or gametocyte (our reproductive cells) the change can be passed down to your offspring without affecting you. However, Genome editing has a long way to go before it could be used as a therapeutic agent.
Thanks for that i have one more question? Is it possible crispr will work for autoimmune diseases if certain genes are identified? Such as rheumatoid arthritis, ankylosing spondylitis, chrons disease etc?
Sikh Unit Yes it is indeed possible. This is the promise and potential of CRISPR Cas9; because of its simplicity, several different loci can be targeted at the same time, making it the perfect candidate to gene therapy where more than one gene is responsible for the disease phenotype. Here is a great paper that talks about the potential of gene therapy for autoimmune disorders: www.nature.com/gt/journal/v7/n1/full/3301111a.html
Hi Matt, have you had the chance to view our CRISPR playlist ( th-cam.com/video/1aJxXWkE3Ek/w-d-xo.html ) which covers a wide variety of topics in CRISPR? It includes an introduction as well as actual how-to videos based around real case studies. CRISPR is a complicated topic, but these videos should give you a good starting point! Otherwise, we also have a dedicated knowledge base with more information: goo.gl/df55ap
Hi Chi, if you have Cas9 & gRNA transiently transfected into your eukaryotic cell, they will be present in the cell for several days until cell division or other factors remove the foreign materials. Otherwise, if Cas9 & gRNA has been stably transfected, they should be present in the cell indefinitely. This is, of course, also dependent on whether you have a constitutive or inducible promoter for Cas9 and/or gRNA. Hope this helps! Our knowledge base has more info: goo.gl/kCDIbA
Hi there, thanks for your question! We agree that it is important to credit the scientists involved in these fantastic discoveries (and have credited several of the scientists involved in our other CRISPR videos). There is a lot of material covering the history of CRISPR, I can give you a brief overview: the history of CRISPR begins in 1987, when Yoshizumi Ishino from Osaka University discovered the first CRISPR loci (this was also, thanks to work done by Mojica and Juez from the Unviersity of Alicante as well as Jansen from Utrecht University in the following years). In 2006, NCBI's Eugene Koonin proposed that CRISPR is part of the prokaryotic immune system. All of these discoveries ramped up excitement for CRISPR. Jennifer Doudna generated structures of various CRISPR nucleases and figured out CRISPR's mechanism. Emmanuelle Charpentier identified CRISPR RNAs. Charpentier & Doudna collaborated to isolate and identify Cas9 and how it can generate double stranded breaks in DNA at specific locations, allowing insertion of DNA chosen by the scientist. Feng Zhang's background in using gene editing systems on mammalian genomes enabled him to swiftly enter the scene and humanize/commercialize the tecnique.
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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 to join and we're excited to hear what you guys think of it! Sign up at: info.abmgood.com/crispr-crash-course
Pest bubonic runnnnnn 😨
Check out our newest video about CRISPR/cas9 and the methods and tools available to put this amazing procedure to use! - th-cam.com/video/INC_kdr7I34/w-d-xo.html
If you haven't seen our previous CRISPR/cas9 introduction video be sure to also check it out! - th-cam.com/video/1aJxXWkE3Ek/w-d-xo.html
Thank you ! I think it's a "brief" for experts :) If I hadn't learned about this from other videos before, this would have been some high-level information.
But when I saw this detailed series, you cannot imagine how I got excited.
Have you seen our video on the methods and tools available for using the CRISPR Cas9 system in your research? - th-cam.com/video/INC_kdr7I34/w-d-xo.html
Great video! If I wanted to knock out a gene in C. diff by using bacteriophage deployment of the cas9 protein, complementary gene sequence, and the guide rna, how would I do so?. A few more questions are:
1. Which of the 3 viral delivery methods would be bacteriophage?
2. How would I go about designing the dna strand that I want to insert?
3. How would I get the edited bacteriophage to grow clones?
4. How can I assure that the bacteriophage will target the C. diff?
5. How can I tell if c. diff uses the Nhej method or the hdr method of dna repair?
6. What can I use to find the base sequence of a particular gene in the c. diff genome?
Sorry for all of the questions!! I hope that you all can help 😁
Hello there! Thank you for the comment! We would love to answer your questions at technical@abmgood.com.
Hello abm, I've been enjoying your videos on CRISPR and, if you have the time, I would be very grateful if you could answer a few questions I have on the terminology used!
1. What is a primary cell?
2. Why can adenoviruses affect primary cells and not lentiviruses?
3. Other than dividing/non-dividing/primary cells, what other types of cells are there?
4. What does 'differential tropism' mean?
5. How does differential tropism make AAV a candidate for regenerative medicine?
Thank you for your patience.
Hi Natsukashii,
Thanks for watching our video! To answer your questions:
1. Primary cells are cells that are collected directly from living tissue and then grown in vitro. Scientists may prefer using primary cells for their research because they their characteristics are close to how the cell actually behaves in its native environment (as opposed to immortalized cells that have undergone so many population doublings that they may have altered characteristics).
2. Both adenoviruses and lentiviruses have the ability to infect many cell types, both dividing and non-dividing. The adenovirus, however, has a higher transduction rate towards primary cells.
3. Primary cells can fall under dividing or non-dividing cells depending on what cell you are looking at. For instance, neurons are considered non-dividing cells as they are fully differentiated. On the other hand, glial cells and other supporting cells of the brain do have the ability to replicate and are thus considered "dividing cells". Aside from Primary cells, other cell types are immortalized cells, stable cells, and stem cells.
4. Differential tropism likely refers to the idea that certain viruses can infect certain types of cells/tissues. For example, AAV has many serotypes (variations) that enable it to infect a broad range of tissues whereas other viruses like the rabies virus primarily infects neuronal tissue.
5. Because different variations of AAV have a capacity to infect different tissue types, gene therapies can be delivered to very specific locations or to most all tissue types, depending on the need. For example, AAV Serotype 2 has been used in cancer treatments to kill a diverse group of cancer cells without showing any effect on healthy cells. There's more information about AAV on our knowledge base if you'd like to learn more: goo.gl/F7lvrL
great explanations, Thank you!
Vitalie Samoil Glad you found the video helpful! If you're interested in learning more, we have an additional video in the CRISPR series on gRNA design (th-cam.com/video/dXPDefej0Ps/w-d-xo.html).
Hello. I am working with CRISPR to delete a functional gene from Salmonella.
Once the gRNA has been designed, is it possible to include them along with the Cas9 enzyme in a plasmid before being transfected into the Salmonella ?
Hi Najwa! Thanks for watching our video and thanks for leaving a comment. Yes, it is possible to deliver both the sgRNA as well as the Cas9 in the same plasmid to be transfected into your target organism/cell. At abm, we offer "All-in-One" plasmids that can do the trick! May I suggest you take a look at our Genome-wide sgRNA Library to see if we have something of use for you? goo.gl/RpdVZb
These non-viral methods talk about the insertion of RNA into the in-vitro cell, or can those chemical transfections be done inside a living animal? (If so, on what scale? A single tissue, a single organ, or a full organism?) And are these methods (both viral and non-viral) good for the RNA insertion and Cas9 protein as well? Or is the Cas9 inserted in a form of RNA and the cell produces the Cas9 protein from the inserted RNA?
Hi there, thanks for watching our video! Yes, CRISPR Cas9 can be used to modify the genome of a living animal -- this can be done on a tissue-specific level (using viral gene delivery methods like AAV) or to an entire organism (creating transgenic organisms by injecting CRISPR/Cas9 machinery into the embryo). The methods described in the video are commonly used to delivery either the sgRNA, the Cas9 protein, or both into the target cell -- it depends on what you'd like to achieve. For instance, if you'd like to have the cell stably express Cas9, you could use a lentiviral delivery of the Cas9 gene to stably integrate the gene into your cell's genome, allowing it to constitutively express Cas9. Then, all you'd need to do to begin genome editing is to provide the sgRNA targeted to your gene of interest (you could do this by delivery of the sgRNA plasmid via electroporation for transient genome editing). If you'd like more information about how to plan your genome editing experiment, visit our knowledge base: goo.gl/df55ap
If I understand correctly your response, you are saying that you can deliver the Cas9 protein in the lentivirus? (I'm confused by two opposed meaning sentences in your explanation above.) Isn't that too large for the virus or less efficient than using RNA form of the Cas9? I thought the problem with the CRISPR Cas9 is that we have not enough space in the delivery vesicles. Can in-vivo cell create Cas9 out of a delivered version of Cas9?
Hello!
I really enjoy your videos about CRISPR/Cas9!
I already heard about this method. People say that it's controversal because it would be difficult to tell if there's a GMO or not. In my country, germany, GMO's are under strict conditions. But if you transfer the coding sequences for the Cas9 protein and the gRNA in the target cell, it should be obviously an GMO.
So why is it so hard to tell if it's a GMO or not?
Hello Tony! Thank you for watching and leaving a comment! The debate is centered around the difference between what it means to be "genetically modified" vs. "gene-edited". To make a "genetically modified" plant, for example you would add a gene from another organism to that plant (e.g. adding a gene from a bacteria that helps corn be resistant to drought). To make a "genetically edited" plant, you would be editing the plant's existing DNA in order to improve a characteristic of the plant (e.g. deleting a few nucleotides in the mushroom's DNA so that it doesn't turn brown as quickly). Current regulations only include the definition "genetically edited" and therefore so far, in the USA, at least, some CRISPR gene-edited plants have been given the green light to be grown and maybe even sold without too many regulations in the way.
Also, there are many ways to do gene editing in an organism without needing to incorporate the CRISPR Cas9 gene into the cell itself! You can transiently deliver Cas9 and gRNA to temporarily edit the cell before the cell gets rid of it naturally. See our knowledge base for more details: goo.gl/df55ap
well explained.
can you please make a video on use of CRISPR cas9 for functional analysis of gene in filamentous fungi.
Thanks for your suggestion! That sounds like an interesting topic to cover.
I have read paper with AAV viruses that are delivering 5.1kB CRISPR constructs, why does your video say the capacity is 3.4kB?
Hi Tt Cooper, thanks for watching and commenting! Yes, it is possible to package a 5.1 KB construct. With AAV, the success of packaging large constructs >4.4 kb is sequence specific and can often result in a significantly lesser yield. We mention 3.4 KB as the packaging capacity as, in our experience this does not compromise yield.
I will cry!
The cas9 from S.aureus and S.pyrogenes show infectious diseases in humans,so how can we redesign cas9 or which bacterial protein will be suitable other than s pyrogenes and s aureus ..in order to overcome this issue.
Hello!
Thanks for the wonderful information.
I have to write a research proposal - Crisper based mouse model for cancer theraputics.
Can you please guide me.
Is the CRISPR/cas 9 system delivered in the form of DNA to the target cell?
vasco cluny the most commonly used method relies on using viral vectors to deliver the Cas9 gene and the gRNA sequence to the target cell which is then expressed. However, it is also possible to induce the target cells to take up the Cas9 protein and gRNA sequence (though the efficiency of the transformation will be much lower) - abm offers pre-design viral vector constructs for the Cas9 proteins and a wide range of gRNA sequences: www.abmgood.com/CRISPR-Cas9-sgRNA.html
thank you very much!
Hello! Thank you for these videos, they are great! though I have a question..
Is it possible to introduce the complete crispr-cas 9 protein with sgRNA attatched to a target cell instead of the genetic material that will be transcribed and translated into the final protein form?
I am attempting to use crispr-cas 9 to knock out a hox gene (CDX 1) in a zebrafish zygote. The ideal situation would be to have the complete Crispr cas 9 protein with bound sgRNA already present in the egg, in an inactive state, and have it activate at fertilization. If I introduce this endonuclease machinery as genetic material to be translated to protein, I suspect that developmental cleavage of the zygote will have begun by the time the crispr protein is translated, leading to only some of the blastomeres will be acted on by Crispr.
Hi Alejandro, thanks for watching and leaving a comment! I believe the CRISPR technology you are referring to are the Cas9-sgRNA ribonucleo-protein complexes (RNPs). I don't believe there is currently a well-documented way of keeping this complex dormant inside the egg until you need to activate it. However this complex begins work quickly after transfection so you can deliver the RNP complexes via microinjection/electroporation to the egg right after fertilization, a common technique that is used to deliver RNPs. We do offer custom CRISPR RNPs, if you are interested -- send us an email to learn more: info [ at ] abmgood.com!
Hello, I may have misunderstood something, but I'm a bit confused. All these methods are described as being used to introduce genes to the cells, but Cas9 is a protein. Do these methods introduce teh cas9, guide RNA, and the gene you are introducing, or only the latter?
It all depends on the delivery method used when performing knock in experiments, where the presence of Cas9, sgRNA and desired repair template (this could be a whole gene or a single nucleotide mutation) are required.
Generally speaking, there are two main ways to introduce the components mentioned above for gene editing (insertion of gene or making a mutation in a gene, but not knockout):
- Non-viral or non-integrating: This typically is known as the more transient way, where Cas9 is introduced into the cell as a protein, mRNA, or expressed from a plasmid, while the sgRNA is introduced as RNA or expressed from a plasmid. Alternatively, these can also be delivered by a non-integrating virus like AAV or Adenovirus. These are known as non-integrating methods, so once the genomic edit is made in the host cell, the Cas9, sgRNA and repair template shouldn't persist in the cells after several rounds of cell division.
- Viral integration: When using integrating viruses such as lentiviruses, the Cas9 and sgRNA sequence are delivered into the cell via viral RNA and usually integrated into the host cell genome along with the rest of the viral backbone sequence (the repair template can be provided separately via transfection or can be included in the viral payload). This will allow persistent expression of Cas9 and sgRNA for a higher success rate for editing, and will also allow for selection of infected clones if a selection marker such as puromycin is included in the viral backbone. Some experiments may not prefer the persistent expression of Cas9 and sgRNA once the editing has taken place, so in these cases, we would recommend going with a non-viral or non-integrating method.
Hi! Thanks for making these videos. I’m new to this technology and I’m trying to understand how it works in real life situations. What I still don’t understand is if I can introduce Cas9 and a gRNA using ONLY a plasmid, even if I’m working with human cells, or if this is the case, can I use ONLY a virus or electroporation for example, to deliver Cas9 and the gRNA?
Thank you for your question and we're glad you're enjoying the videos. If you are working with human cells, you have several options, and we will elaborate on the most common ones for each of the methods you mentioned:
1) DNA Transfection: You can use a DNA transfection reagent to deliver Cas9 and the gRNA to the cell. The Cas9 and gRNA can be supplied on two separate vectors, or can be on the same vector, typically called an All-in-One system. This method highly depends on whether your cell type has a high transfection efficiency.
2) Electroporation: You can also electroporate plasmids into the human cells, but more commonly, scientists use electroporation to deliver recombinantly expressed and purified Cas9 protein with transcribed gRNA. This is typically called a RNP (ribonucleoprotein), though electroporation effectiveness can highly depend on cell type.
3) Virus: This is one of the most effective methods of Cas9 and gRNA delivery, as long as your cell type is susceptible to viral transduction. The virus can be a two virus system (i.e. one virus carrying the Cas9, one virus carrying the gRNA), or it can also be an all-in-one system (one virus carries both the Cas9 and gRNA).
If you want to learn more about how to use viruses to deliver Cas9 and gRNA to mammalian cells, you can visit our Case Studies page here which walks you step by step on how a group of researchers used our All-in-One Lentiviruses to achieve mouse cell knockouts:
www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Case_Studies.php#Knockout
Can I transfer crispe cas through agrobacterium in plants
Hi Nazima, thanks for your comment! Argobacterium delivery of CRISPR/Cas9 into plants has been demonstrated -- there's a paper here you can read on one such experiment: goo.gl/6kCtYj
please I wanna know more about the off target of both crispr cas9 and dcas9
+Amna J Hi Amna! Thanks for asking - may I refer you to our knowledge base where we discuss off-target effects? ow.ly/4mHJnY
Thank you!
hi this is Arti. I want to learn this technology for my research work in Algae, so can you suggest me the way how actually this technique works in real time.
Hi Arti, thanks for leaving a comment. We currently don't offer any resources/videos on using CRISPR in plants. We do have "how to" videos demonstrating how to use this technology in mammalian cells (see: How to perform a CRISPR Knockout Experiment - th-cam.com/video/1BSiFfyObTY/w-d-xo.html, or How to perform a CRISPR Knockin Experiment - th-cam.com/video/PlVy1oupFxw/w-d-xo.html), and in bacteria (see: How to perform a Bacterial CRISPR Cas9 Knockout Experiment - th-cam.com/video/7pOR3ssg7dg/w-d-xo.html). I hope this helps.
Hi I want to use this for my research by using phagemid.Can you please help me regarding that how I can use
Hi Tasnuva! If you want to learn more about this topic, I recommend that you check out our CRISPR knowledge base article which covers more in-depth information: old.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Tools_Methods.php
Don't we only need to let the sgrna in ?why are we talking about dna entering ?
and finally how can we let the cas9 enzyme in?
wish to respond fast.thank you
Most commonly, the sgRNA and Cas9 are delivered into the target cell as DNA sequences on a plasmid. The cell's machinery then performs the transcription to generate the sgRNA, as well as the transcription and translation for the Cas9 protein. The delivery of the genetic DNA sequence can be done using one of 2 ways:
1) DNA Transfection: You can use a DNA transfection reagent to deliver Cas9 and the sgRNA to the cell. The Cas9 and gRNA can be supplied on two separate plasmids, or can be on the same plasmid, typically called an All-in-One system. This method highly depends on whether your cell type has a high transfection efficiency.
2) Virus: This is one of the most effective methods of Cas9 and sgRNA delivery, as long as your cell type is susceptible to viral transduction. The virus can be a two virus system (i.e. one virus carrying the Cas9, one virus carrying the sgRNA), or it can also be an all-in-one system. The viral payload (carrying the Cas9 and sgRNA sequences) then gets integrated into the host cell genome (in the case of lentivirus) or gets transcribed directly from transient DNA-genome viruses such as adenovirus or AAV.
A third way that doesn't require the use of DNA is Electroporation. You can also electroporate plasmids into the human cells, but more commonly, scientists use electroporation to deliver recombinantly expressed and purified Cas9 protein with transcribed sgRNA. This is typically called a RNP (ribonucleoprotein), though electroporation effectiveness can highly depend on cell type.
If you want to learn more about how to use viruses to deliver Cas9 and sgRNA to mammalian cells, you can visit our Case Studies page here which walks you step by step on how a group of researchers used our All-in-One Lentiviruses to achieve mouse cell knockouts:
www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Case_Studies.php#Knockout
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Thank you
But won't the host cell reject the foreign plasmid or even refuse to transcribe it?
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It is unlikely that cells will reject the plasmid once it is successfully transfected into them. Transfection efficiency and being able to get the DNA into the cells usually plays a larger role in this case, and each cell type responds differently to various transfection reagents and delivery methods (eg. some transfection reagents could be more cytotoxic to some cells, other cells are not as susceptible to chemical methods (transfection reagents) vs. biological methods (viruses)).
Once in the cell, the promoters used in the plasmid do play a big role in controlling transcription. You have to make sure the promoters in the plasmid are active in the host cell type you are working with. For example, some promoters are constitutive and active in most cell types, while others are tissue-specific promoters and are only active in certain cell types. Some suppliers (including abm) offer reporter vectors or promoter blast kits where a collection of plasmids with promoters driving a reporter like GFP or RFP can be used to transfect cell lines of interest to find the promoter that is most active in a particular cell line. The best promoter is then selected for use in the plasmid that will express the researcher's gene of interest.
As with all experiments, other factors could be involved in transcription and translation once the DNA gets into the cells, so it is always best to verify expression by RT-qPCR or RT-PCR, or by Western Blot when possible.
Can crispr edit genes while you are alive or is it your offspring who only benefit for example if i have a genetic disease and crispr disables that gene will that mean i will be cured as well as my off spring?
The question that you have brought up has many intricacies that needs to be taken into consideration. Most of what we know today about genome editing is from work done on cultured cells. Looking at cells grown in culture, when the genome is altered, that altered genome will be passed down to their offspring.
There's a lot more involved when it comes to humans. theoretically, changes made to a somatic cell will only affect the cells involved and therefore the paitent involved, however, if the change is made to gamete, germ cell, or gametocyte (our reproductive cells) the change can be passed down to your offspring without affecting you.
However, Genome editing has a long way to go before it could be used as a therapeutic agent.
Thanks for that i have one more question?
Is it possible crispr will work for autoimmune diseases if certain genes are identified? Such as rheumatoid arthritis, ankylosing spondylitis, chrons disease etc?
Sikh Unit Yes it is indeed possible. This is the promise and potential of CRISPR Cas9; because of its simplicity, several different loci can be targeted at the same time, making it the perfect candidate to gene therapy where more than one gene is responsible for the disease phenotype.
Here is a great paper that talks about the potential of gene therapy for autoimmune disorders: www.nature.com/gt/journal/v7/n1/full/3301111a.html
so how do you use crispr on a chicken,can you tell me how to do each step from the beginning,please i might be able to change the world
Hi Matt, have you had the chance to view our CRISPR playlist ( th-cam.com/video/1aJxXWkE3Ek/w-d-xo.html ) which covers a wide variety of topics in CRISPR? It includes an introduction as well as actual how-to videos based around real case studies. CRISPR is a complicated topic, but these videos should give you a good starting point! Otherwise, we also have a dedicated knowledge base with more information: goo.gl/df55ap
thanks
I taught a neighbor boy how to make an electroporator from an AM FM radio powered by 9 volt batteries.
Sounds cool!
Greetings! I have 2 questions: 1) Can Cas9 exist in an eukaryotic cell for a long time? 2)Are sgRNA and Cas9 easily degraded? Thank you!
Hi Chi, if you have Cas9 & gRNA transiently transfected into your eukaryotic cell, they will be present in the cell for several days until cell division or other factors remove the foreign materials. Otherwise, if Cas9 & gRNA has been stably transfected, they should be present in the cell indefinitely. This is, of course, also dependent on whether you have a constitutive or inducible promoter for Cas9 and/or gRNA. Hope this helps! Our knowledge base has more info: goo.gl/kCDIbA
We can use this to rid Covid19.
The Broad Institute has actually been actively developping protocols for detection of COVID-19 using CRISPR diagnostics!
Who discover this? If this technology is so amazing why not credit the scientists that discover it? I know Google will tell us
Hi there, thanks for your question! We agree that it is important to credit the scientists involved in these fantastic discoveries (and have credited several of the scientists involved in our other CRISPR videos). There is a lot of material covering the history of CRISPR, I can give you a brief overview: the history of CRISPR begins in 1987, when Yoshizumi Ishino from Osaka University discovered the first CRISPR loci (this was also, thanks to work done by Mojica and Juez from the Unviersity of Alicante as well as Jansen from Utrecht University in the following years). In 2006, NCBI's Eugene Koonin proposed that CRISPR is part of the prokaryotic immune system. All of these discoveries ramped up excitement for CRISPR. Jennifer Doudna generated structures of various CRISPR nucleases and figured out CRISPR's mechanism. Emmanuelle Charpentier identified CRISPR RNAs. Charpentier & Doudna collaborated to isolate and identify Cas9 and how it can generate double stranded breaks in DNA at specific locations, allowing insertion of DNA chosen by the scientist. Feng Zhang's background in using gene editing systems on mammalian genomes enabled him to swiftly enter the scene and humanize/commercialize the tecnique.
Great. Thanks!