hello guys,
I have been struggling to design a gRNA , yes there are tons of materials out there, many website but honestly I cannot begin to say which ones give me a better options or not.
So let's say I download the mRNA from NCBI for a gene, what should I do next? if anyone could give a step by step way to do it, I appreciate it
Thanks
https://portals.broadinstitute.org/gppx/crispick/public
I think it can be a waste of time trying to decide which sgRNA predictions are better than others. It seems this will be dependent on many factors and design tools can only cover the gap so much. If you have something specific in mind, then maybe a certain tool is better suited. But if your goal is to simple design an sgRNA to disrupt a gene, then the broad portal linked above should do a good enough job. I think the key is to test the guide as soon as you can.
Steps for Broad tool: 1) Select a genome (specific version shouldn't matter very much) 2) Mechanism: CRISPRko is the "normal" one. 3a) Enzyme: SpyoCa9 (NGG) is the "normal" one. 4b) select Chen or Hsu. You can check which type of tracrRNA you will use, but I'm not convinced this is too important. 4) Insert target gene name and select it. 5) Click validate and fix errors if needed. 6) Click submit from the new summary page. 7) Download results and clone sgRNA sequence into your sgRNA vector.
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Seconding this. Just load your target sequence into any of these predictor tools and use what they return. If it is human you might also check the GeCKO library (for example at Addgene) which contains many gRNAs for the human transcriptome. After all, whether a sequence works or not is just a prediction, and I heard talks where a given sequence worked well in one celltype and not at all in another one, so it's in the end a lot of trying and screening work.
@rfran010 thanks a lot, it is very helpful, one stupid question, lets say I cut a gene using Cas9, do you know how the cas9 affect a gene? is it only binding to a PAM region then cut the 3 left side nucleotides? or is it random cut etc ? do you have any reference or any knowledge on this?
This question, will all due respect, shows that you have done no reading on CRISPR-Cas9 at all. It's the very basic of how it works, and you can even answer that with the Wikipedia article.
I agree this is very basic CRISPR mechanism and you'd be better off reading sites that could explain better than me. However, there is a lot of information out there and I figured from the initial question this was all very new to you, so I'll try to leave a couple guiding marks below.
There are many CRISPR-based systems. The "default" CRISPR system is one that uses Cas9 and sgRNA to target a region and cut it. These elements are pretty much universal to all CRISPR systems, but different systems will have variations on how each element works specifically. In the simple system, sgRNA acts as a guide for the Cas9 enzyme. Through base-pairing of the targeting sequence, Cas9 finds its target, then cuts. The PAM site, essentially, simply defines which regions in the genome are allowed to be targeted. The cut site is defined relative to the PAM site. After cutting, to disrupt the gene you rely on something going biologically wrong (i.e. improper repair of the cut site). These errors can results in mutations where a frameshift mutation can disrupt the whole gene. After reading, you'll see there are many different approaches that hope to accomplish different tasks or more specific tasks, including a strategy to more specifically delete a region using two sgRNAs that target the two regions around the fragment you want to delete.