Tips for Successful CRISPR Research

Sharing is caring!

Over time, there have been many types of genome editing tools gracing the world. Tools like Tale Nucleases and zinc Nucleases had their popularity, but they were primarily complex tools to handle. However, CRISPR Ca9 came into the gene-editing scene in 2012 through Emmanuelle Charpentier and Jennifer Doudna. CRISPR is a more straightforward tool to use; many compare this tool to a scissors-like tool. As easy as the CRISPR tool seems, it has its complexity. Here are some easy tips you can employ;

• Determine the edit you’re trying to make

The first step you should take when using the CRISPR Cas-9 is to know what you’re editing. There are many things you can do with a gene-editing tool. You can replace a gene totally or edit the gene. When you determine what you want to do with the need for the gene-editing tool, you can start with the experimental design. After that, you can begin to navigate your gene-editing tool to suit your needs.

• Get an editing strategy before you start.

If you want to use the CRISPR tool, it’s very easy. Where you may have an issue is discovering the suitable genotype. There are different methods you can employ to get an appropriate genotype. Some of these methods include; 

1. Sanger Sequence: This method depends on the sequence of the PCR sequence. 
2. Illumina Sequencing: This sequence is similar to the danger sequence but has better output. 
3. Pcr detection: this method depends on gene detection to delete and detect the genes. 

• Choose Your PAM Site

When you want to edit your gene with the CRISPR editing tool, you need a Pam site. This Pam site has to be close enough to your editing site to help you increase the accuracy of your gene editing. For instance, if you’re trying to edit a protein gene, all you need to is try editing the initial exons with your tool. Moreso, if you want to knock out an Sp. Cas 9 protein, the closest PAM site you’ll have is the NGG PAM site. 

• Design the RNA

To use a CRISPR Ca9 editing tool, you’ll need to understand how to use the RNA tools. There are two major types of RNA that work with the Cas 9 gene-editing tool. The first RNA is tracrRNA which acts as the structural tool at a 3′ end. The second RNA is the crRNA; this component connects the CRISPR tool to the targeted gene. This second component is always located around the 5′ end. You also have to choose the type of RNA that works well for the editing you wish to carry out.

• Get your HDR Template

When you don’t want any regular gene editing, you may want to make a particular change. In this case, you have an HDR Template that should help you make the accurate change. If you’re going to use the HDR Template, you have to activate the template when you’re preparing the edit site. 

• Know Your Cells

If you want to edit your genes excellently, you need to understand the cells you’re dealing with. This will help you use the CRISPR editing tool to the highest transfection efficiency. Once you get the transfection efficiency well, the editing has no option but to come out excellent. When you check your cells and determine their type, you have the liberty to choose the best possible method to edit the genes. 

• Positive Control and Checks

When you edit genes, you have to check how accurate the editing has been. You can start with a transfection control to the reagent function. When you’re done checking the positive control, the next step is to check the accuracy of the gene editing. A great technique you can use to check this editing efficiency is to use the cleavage detection kit for gene editing.

Courtney P.

I’m a 20-something stay-at-home mother and wife. I have an amazing husband, a beautiful daughter, two loving dogs, and a lazy cat. I wouldn’t change my life for anything! I love to read, listen to music, cook and blog!

Sharing is caring!