You must check out this highly-recommended gRNA design tool | Ubigene

As long as you know a little bit about CRISPR/cas9 technology, you must hear that during CRISPR/cas9 experiments, the design of the gRNA will largely affect the success rate of the project. The cas9 nuclease, under the guide of gRNA, targets the gene of interest and generates a double stranded DNA break (DSB). Insertions and deletions (indels) arise as a result of random repair of DSBs, causing frameshift mutations that result in premature stop codons, this process can achieve the purpose of gene knockout. But in most cases, many people often face difficulties at the stage of gRNA design. So this week we’ve also received some questions about gRNA design and selection, let’s start our discussion:

Q1: While we design the gRNA to KO the target gene using CRISDPR/Cas9 technology, should I use single gRNA or dual gRNA? What is the difference between them? A: In theory, a single gRNA can target only 1 site of a gene, and the number of transcripts that can be knocked out is limited. While a dual gRNA can largely solve the problems, and can also KO a larger fragment of gene, so the KO effect of the dual gRNA vector will be better than that of the single gRNA vector. But in practice, designing an apporpriate strategy depends on specific cases and analysis. To get the desired experimental results, we currently use the Red Cotton™ CRISPR gene editing system (Go) for gene KO strategy design. This powerful system integrates experimental data from more than 5000 successful cases of gene editing with several mainstream gene databases and it provides 3 different gene KO strategies for free by just entering the gene name. It can reduce our experimental trial and error costs. Q2: What are the principles when designing the gRNA? Could you recommend a useful gRNA design tool? A: You should take many factors into consideration when designing the gRNA, here are the three most important rules you should consider: The sequence specificity of the gRNA should be strong enough gRNA sequences to try to cover as many transcripts as possible Avoid target regions that encode amino acids too close to the N- or C-terminus of the protein But in fact we usually do the design of gRNAs with the help of the gRNA design tool. A good gRNA design tool can improve efficiency and reduce the trial and error cost. I recommend using our Red CottonTM system - gRNA design tool with the help of the sgRNA design website by Zhang’s lab. But just a reminder, it is not the higher score of gRNAs, the better its KO effect. Before you make the decision, you can have a comprehensive knowledge of the target gene using our Red Cotton system. Q3:  How can I improve the targeting efficiency of gRNAs? A: How to improve the targeting efficiency of gRNAs is a matter of great concern to every researcher working on CRISPR/cas9 technology. Either sequence optimization of the gRNA, changing the delivery method of the gRNA vector, or increasing the number of gRNAs used, etc., can improve the targeting efficiency of gRNAs to some extent. Hence, there are many ways to improve the targeting efficiency by optimization according to the actual situations. Welcome to use our free Red Cotton™ CRISPR gene editing system (Go) or feel free to contact us if you need help!

Above are the Q&A of this week. If you have any similar questions, feel free to contact us now!   Ubigene makes genome editing easier!