How to perform a CRISPR Knockout Experiment
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- Опубликовано: 20 мар 2017
- Due to CRISPR's unparalleled ease-of-use and affordability, gene knockout experiments are now more feasible than ever before!
In this video, we will show you how we used the CRISPR Cas9 system to knockout the LIF gene in a colon carcinoma cell line. This is accomplished by the delivery of a Cas9 and sgRNA lentivector into the cell line followed by drug selection and monoclonal isolation. More importantly, we demonstrate how to characterize and verify your CRISPR Knockout using the PCR-based Surveyor Assay, Sanger Sequencing, and Next Generation Sequencing. This complete process of validation ensures that your final CRISPR Knockout cell line is 100% successful.
At the end of the video, we will introduce you to abm’s entire suite of CRISPR Knockout services - designed to help you jumpstart your Knockout experiments!
Read our complete CRISPR Knockout Case Study here:
➜ info.abmgood.com/crispr-cas9-...
Want more tips, tricks, guides, and protocols on how to knockout a gene using CRISPR? Join our free, 4-week #CRISPRCrashCourse: info.abmgood.com/crispr-crash-...
Read more about the basics of the CRISPR system on our knowledge base:
➜ info.abmgood.com/crispr-cas9
Learn more about our CRISPR Knockout Cell Line Generation Service here:
➜ goo.gl/EpEZDB
Watch more videos about CRISPR:
➜CRISPR Cas9 - Gene Regulation with dCas9: goo.gl/tRkHWd
➜ How a mini CRISPR Cas9 system is revolutionizing gene therapy! goo.gl/njRPv2
Thank you to all of the amazing community contributions towards translating this video into other languages including:
➜ Spanish
(To view the translations, toggle the CC button and then go to Settings ➜ Subtitles/CC to select a language.)
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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
Yes, I want learn Crispr-cas9 protocol for genetic polycystic kidney disease called pkd. Pkd called also faulty genes maybe in DNA thanks. I am pkd patient
In an ironic and funny twist of fate, learning to code is actually the most important task of mankind right now. Thank you so much for sharing your insight and knowledge.
Just a clarification, is T7 Endonuclease a surveyor enzyme?. Do you guys mix the mutant DNA with that of the wild type to create hybridization? I am quite new to this topic but I am eager to learn.
Hi Reo,
T7 Endonuclease is very similar to Surveyor enzyme, and they can be used in the same way to detect indels. When screening polyclonal pools it isn't necessary to mix the mutant DNA with WT, since the pool likely contains many different sequences, which will create the mismatch the enzyme recognizes.
However, if you are screening a monoclone, it can be a good idea to mix in WT DNA. This is because there is a chance that both alleles of the gene happened to form the same indel. In this case, if WT DNA wasn't mixed in, the Surveyor/T7E1 enzyme wouldn't cut, and you would have a false negative result.
If you'd like more details, I'd recommend you check out our Knowledge Base article on the subject of CRISPR screening and validation: www.abmgood.com/marketing/knowledge_base/CRISPR_Cas9_Screening_Validation.php#MCDA
Appreciated! Thank you so much!
@@abmgood
@@Jackmotoniwaqa You're very welcome :)
Can this technique work same on humans
What does colony mean here? Like you said, colony 3 and 6 show editing.
Hello Huzzatul! Thanks for watching and leaving a comment! Colonies in this case refers to a cluster of identical cells (called clones) that are derived from a single parent cell! We had to perform a round of antibiotic selection to ensure only colonies of cells that have successfully "taken up" our vector (containing the antibiotic resistance gene) survive. From these clones, we select several for further analysis to check if there was a biallelic knockout in any of them. Hope this helps!
How do you create a nuclear gRNA from NCBI and get it to cut the DNA of interest
If you are asking about how to design sgRNAs from NCBI sequences, we do have another video specifically covering sgRNA designs here: ruclips.net/video/dXPDefej0Ps/видео.html
Briefly, the NCBI input you need depends on the sgRNA design tool you are using. For example, if you use CHOPCHOP, you will either need to use the NCBI RefSeq Accession Number, Gene ID, ENSEMBL ID or the coding sequence itself in FASTA format as input.
Let's take the Human A1BG gene as an example. If we wanted to design sgRNAs against hA1BG, then we would use its RefSeq Accession Number as input, NM_130786.
The design software takes into account off-target mismatches, GC content, target position, and predicted efficiency into account when ranking sgRNA designs. It is important to remember that these are only predicted measures, and it is always best to design more than one sgRNA per gene target to increase your chances of obtaining a successful knockout.
Oops ... thought this was popular-science-oriented.
- goes way beyond the other CRISPR-CAS9-cRNA-TracRNA-PAM videos I had been enjoying.
Hello Gregory,
Thanks for watching! We are really glad to hear that you enjoyed the video :) Please stay tuned for upcoming videos too!
Can several sgRNA templates be cloned into a single plasmid you've mentioned at 1:50?
Yes, this is called a multiplex sgRNA assembly system where multiple sgRNAs are cloned into the same plasmid. We have successfully synthesized multiplex plasmids before and we will be happy to look into synthesizing one for you. Simply inquire at info@abmgood.com.
Hello, is the online CRISPR course still available, i am trying to sign up but it is not working
Hi Souad! Yes the course is still available - perhaps our email has ended up in your spam or junk folder? If you still cannot receive our course emails feel free to contact team@abmgood-email.com and we can re-send the course to you.
Thank you for replying
I tried with my gmail and it did not work then i tried with my universitys email and i works
Can the course be taken by anyone? Is there a need for prereqs?
What is the purpose of doing next-generation sequencing?
Hello Jigme,
NGS can help researchers in many different ways and the purpose may vary depending on the project you are working on :)!
For more details, please try this link: www.illumina.com/science/technology/next-generation-sequencing.html
Thank you!
I would like to cure schizophrenia, but when will gene therapy be realized?
Hi, this article from NIH gives a good overview of where gene therapy is at right now in the US: ghr.nlm.nih.gov/primer/therapy/availability
キレイカラダ first you have to keep dreaming. And then the answers will come.