The CleanNA magnetic bead based kits and reagents are used in many applications. Probably one of the most interesting applications at the moment is CRISPR-Cas9. This revolutionary technique enables numerous of researchers to perform cutting-edge experiments with an unbelievable precision within living cells. The use of CleanNA magnetic bead based reagents in the CRISPR-Cas9 technology is explained by taking the process and explaining the added value of the CleanNA products step-by-step.
The history behind CRISPR-Cas9
Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR are discovered in 1987 in Japan. The involved researchers were working on a specific gene in E. Coli and discovered the repetition of the same sequence of DNA over and over again. When it was discovered that this repetitive sequence plays a role in the immune system, the CRISPR evolution had started. The CRISPR-Cas9 technology is applied in 2012 by researchers of the University of Berkely as a tool for genetic modification.
The technology has been widely covered by researchers all over the world, resulting in many publications, applications and new developments. CRISPR-Cas9 allows researchers to edit the genome by adding, removing or altering specific sections of a DNA sequence. A basic search on Google Scholar for “CRISPR-Cas9” nowadays results in nearly 60.000 hits of which 11.000 are from 2017, showing the enormous amount of attention on this technique by scientists.
The CRISPR-Cas9 process
- Design and synthesize gRNA
- Transfect cells
- Determine frequency of successful edits
- Establish single cell clones
- Screen clones for edit
- Characterize successful edits
Guide RNA (gRNA) is a small piece of synthesized RNA. The guide RNA (also called gRNA or sgRNA) consists of two parts, the target RNA and the tracer RNA part. The target RNA sequence will help guide the gRNA/Cas9 complex to its complimentary target sequence on the DNA in the cell. The tracer RNA provides the information on where to cut to the Cas9 complex. Designing and synthesizing the gRNA is a very specific task and varies per application.
The Cas9 enzymes act like a pair of scissors in the cell. When the gRNA+Cas9 complex is inserted into the cell, the Cas9 enzyme cuts both strands of the DNA. The cell will then recognize that the DNA is damaged and will go forth in trying to repair it. It is this mechanism that scientists use to introduce the changes in the genome.
Examples of a CRISPR-Cas9 workflow
After DNA isolation, the locus of interest is amplified using PCR. To determine the success rate, the PCR products are cloned into a vector and then transfected into bacteria.
Once single clones have been obtained, the Plasmid DNA will be isolated from the bacteria
Sanger sequencing will allow each isolated plasmid sample to be screened for success of the CRISPR-Cas9 experiment.
After the CRISPR-Cas9 reaction, single cell/well plates are created to grow the cells. Once the growth is sufficient, the gDNA is isolated from the cell population.
After the DNA isolation, the locus of interest is amplified using a PCR. To determine the success of the reaction, the samples will be sequenced.
The CleanNA products used in CRISPR-Cas9
The CleanNA paramagnetic bead based kits and reagents can be used through the CRISPR-Cas9 process.
For the isolation of the Plasmid DNA as described in approach 1, CleanNA offers the CleanPlasmid DNA extraction kit. The process is designed and optimized to obtain DNA with a high purity and an excellent yield.
When approach 2 is followed, the gDNA extraction from the cell culture, can be performed using the Clean Blood & Tissue kit. A high yield and purity of genomic DNA is a benefit for the downstream application. The Clean Blood & Tissue DNA kit offers the ideal solution to achieve exactly that.
To increase the reaction purity and completely remove the salts introduced during the purification, the CleanNGS and CleanDTR reagent kits can be used.
The CleanNGS magnetic bead based reagents are used to purify the samples after the PCR has been completed. The resulting samples are of high purity, without the addition of any reagents. Therefor the samples are ready for the Big-Dye reaction and preparation for sequencing. The performance of CleanNGS is excellent due to its unique and well established buffer system and the production of the magnetic beads.
CleanNGS can also be used in gene expression experiments such as 5’ RACE and 3’ RACE. CleanNGS is produced RNase free and this feature makes it into the ideal candidate for purification and size selection in Next Generation Sequencing (NGS) RACE library preparation processes. The RNase free production of CleanNGS enable the product to be used for RNA and cDNA purifications. The CleanNGS has been optimized for excellent size selection performance within the latest library preparation techniques.
After the Big-Dye reaction has been performed, the samples need to be purified again. When using the CleanDTR magnetic bead based kits, the purification does not include any salts and therefor results in a sample with an increased purity. When the sample is injected in the sequencer, the absence of salts will result in a higher efficiency of the injection. The increased efficiency is countered by decreasing the amount of Big-Dye in the reaction setup, resulting in a cost-efficient big-dye reaction setup for Sanger sequencing.
The advantages of CleanNA in CRISPR-Cas9
The CleanNGS kits are produced RNase free without additional costs and offer a fast and reproducible sample cleanup. The CleanNGS products are magnetic bead based and allow for easy automation on nearly any liquid handling platform available in the market today.
The magnetic bead based kit and its buffer components typically result in a high yield and purity, even when used in a high throughput and automated setup. With the increased interest in CRISPR-Cas9 applications and the growing amount of experiments, the need for a high-throughput automated solution increases per day. Due to the speed of the protocol and absence of heating steps, the CleanNA kits provide the ideal solution for DNA isolation and purification in CRISPR-Cas9 applications.
Also read about PCR & Real-Time PCR.