Current CRISPR gene editing is hindered by off-target effects and on-target efficiencies. In this on-demand webinar, hear from Abcam’s gene editing experts, Nicole Ha & Dr. Yongwon Kwon, to discover how the optimization of guide RNA (gRNA) design offers a solution to improve the efficiency of CRISPR editing.
Join our speakers as they explore the challenges underlying good gRNA design and the relevant tools needed to assist with this process, as well as discussing the recent data from Abcam’s novel dual-guide-based method showing up to 100% efficiency in CRISPR knockout.
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YK: No, the dual-guide method does not increase off-target effects. Since the dual-guide method does not require highly specific guides, we can select the guide with more stringent specificity which rescues the high off-target score and number of mismatches. We also use an RNP complex to minimize off-target effects.
YK: This method applies to mouse and human cell lines. But currently, we are testing this method to generate knock-out mouse lines.
NH: With a dual-guide method you can modify different cell lines. With different cell lines, you can use them to generate different animal models. There are no restrictions if you use the dual-guide method.
KE: What I would add is, it is of great importance to find methods that can increase the efficiency of generating knock-out animal models. Currently, there are new papers that are being published where this technology is being used for clinical studies.
NH: The dual-guide method is novel and new to the CRISPR user. It may not be utilized across the industry. Proper design of a dual guide, by focusing on minimizing the off-target effects, is important to achieve high editing efficiency. This is especially important for researchers and companies looking to bring their work to clinical trial stages.
KE: The advantage of the dual-guide method is that it does increase your confidence that you will generate a knock-out. And in the space of drug discovery, it is quite important to have this confidence. So that is where this technology has a lot of promise. To add to that, what comes to mind as well, is how it can be extended to other fields of life sciences. We are excited to see what will come up in the literature within those fields.
KE: There are a lot of applications where CRISPR has proven to be very useful in the health sector, but also in the agriculture sector. For example, CRISPR is being used to provide resistance of crops against pest infection. Another example is CRISPR being used to eradicate mosquitoes that could contaminate a population with malaria. There are numerous examples where CRISPR is involved in different aspects of life sciences research.
NH: I agree, there are many different applications where CRISPR is currently being used. I know that CRISPR is currently also used to develop some of the vaccines for COVID-19. In Abcam, we are developing certain knock-out cell lines to help researchers in this endeavor.
NH: We use Sanger sequencing or next-generation sequencing (NGS) to show that the dual-guide approach has higher cutting efficiencies in the outcome. Because the fragment deletion is between the two Cas9 cutting sites, it is highly favorable. For example, we have got 99% of B2M gene knock-out efficiencies in the A549 cell line that has a 40-base pair fragment deletion showing 99% cutting efficiency. This is evidence that the two Cas9 dual-guide methods cut exactly at the same time.
YK: The optimal size of fragment deletion is from 40 to 200 base pairs.
NH: We use a ribonucleoprotein (RNP) complex to minimize the off-target effect. After the RNP transfections, we compare the knock-out efficiencies of the dual guide with the single guide alone. To analyze the edited and control samples, Sanger sequencing and NGS are performed. Analysis of efficiency using the analysis tool is compared between the dual and the single guide. Knock-out efficiency is represented as a percentage of the sequence in the cell that continues the frameshift, including indels for a frame deletion.
YK: In the case of knock-out, we provide only homozygous loci clones, which are confirmed by Sanger sequencing and NGS.
NH: It does. When you are using the dual guide versus the single guide, you are essentially increasing the cutting efficiencies twofold. This reduces the number of transfection rounds needed to produce the homozygous clone, saving months of work, and reducing the cost of screening multiple clones.
KE: It depends on the field of research. In academia, scientists mostly do basic research that is typically not well funded. Therefore, they work on cell line models most of the time. The labs with better funding have more money to generate animal models. In pharma, however, both models are equally used, especially in drug discovery projects in the early phases. Cell line models are used to validate the target of a drug. In the later stages of projects or the preclinical phase, is where animal models are utilized the most. Therefore, it depends on the field of research. But in the pharmaceutical sector, both models will be used equally.
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