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Designing Crispr guides and cloning into appropriate vectors

molecular cloning Crispr/Cas9 gene knockout lentiviral vectors

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#1 Natalia KM

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Posted 13 September 2019 - 12:59 AM

Hi there,

 

I am new to cloning and have recently carried out some cloning using the Gateway system with a lot of help from some really helpful members in this forum!

 

I have to carry out some gene knockouts now and although I am familiar with Crispr/Cas9 knockouts, I have no knowledge of how to design guides and clone them into appropriate vectors. I had a number of questions I was hoping I could get some help on.

 

1. I am aware that there are numerous methods of delivering the Crispr/Cas9 system into cells but not sure what all he different methods are? Can someone explain to me the various methods. My current lab uses the Zhang lab method with the lentiviral delivery system but I would like to find out about what other vectors/systems people use?

 

2. Can someone help me design guides for my gene of interest/give me step by step instructions on how to do this?

 

3. How do I incorporate the guide into my lentiviral vector?

 

Any help would be greatly appreciated!



#2 OldCloner

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Posted 13 September 2019 - 03:16 PM

Hi again, Natalia,

 

I used to build gene targeting vectors that worked by depending on fairly rare homologous recombination between the plasmid-carried mutant sequence and the endogenous sequence on the chromosome. I'm afraid I never did any CRISPR, however I did do some reading about it before I retired.  There is a lot of information on-line about designing guide RNAs, and various on-line tools for doing optimal designs as well. Here is one example:

 

https://www.takarabi...na-design-tools

 

Other companies have similar educational pages, if you do a search on "guide RNAs" or similar search terms you can find many companies offering design help and plenty of publications on the subject.  That is a good place to start.

 

Hope this helps a little. Maybe a "young cloner" can offer more help!


Edited by OldCloner, 13 September 2019 - 03:18 PM.






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