In this on-demand webinar, hear gene editing experts Dr. Frank He and Dr. Yongwon Kwon detail how the optimization of guide RNA design offers a solution to improve the efficiency of CRISPR editing.
In this on-demand SelectScience® webinar, Dr. Frank He and Dr. Yongwon Kwon, from Abcam, explore the challenges underlying effective 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 which can achieve close to 100% knock-out efficiency.
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YK: No, the dual-guide method does not increase off-target effects. Since the dual-guide method does not require a highly efficient guide, we can select a guide with more specificity, which minimizes the risk of off-target effects. Additionally, we use an RNP (ribonucleoprotein) complex to minimize any off-target effects.
YK: Compared to the single-guide method, the use of the dual-guide method increases the knockout efficiency by over twofold, close to 100%. As such, a few rounds of conception and screening are required to generate a homogenous clone, significantly reducing cost and potentially saving months of work.
FH: Since the dual-guide method is a relatively recent advancement in the CRISPR gene editing field, it may not be widely adopted among biomedical researchers, but we employ it here at Abcam with great effect. And we know that with effective guide RNA design strategies one can utilize the dual-guide approach to achieve very high editing efficiencies. As such, use of this approach can more reliably generate knockouts and that assurance is especially important for biomedical scientists and drug discovery researchers.
YK: The optimal size of deletion is from 40 to 200 base pairs. We have tested several different kilobase pairs, but longer ones showed lower efficiencies.
FH: We typically provide homogeneous clones, which we confirm by Sanger sequencing and NGS. However, if the target gene is essential for cell growth and viability, , we typically provide heterogeneous clones.
YK: As we determine whether or not to use CRISPR gene editing, we utilize a set of tools to design the most optimal guides. To achieve a cellular delivered Cas9 guide, we use a ribonucleoprotein (RNP) complex consisting of Cas9 protein and a targeting gRNA. This method offers several advantages over plasmid-based delivery, including the reduction of off-target effects. Typical knockout efficiency with our dual-method approach is up to 100%. Furthermore, at least 14 amino acids are removed. A concrete portion of that becomes the likely outcome.
FH: CRISPR is widely used in the generation of both cell line and animal models across the life sciences. And applications for CRISPR gene editing in organoids are also now in active development. In the drug discovery industry, cell line models are used to validate targets and explore mechanisms of action, whereas animal models are used in preclinical development.
FH: There are a multitude of applications for CRISPR. The speed and efficiency of the CRISPR/Cas9 system is superior to other genome engineering techniques. There are many laboratories today working on method development and broadening the scope of potential applications, and it's such an exciting field to be in. CRISPR/Cas9 is widely used in biomedical research laboratories and research areas such as oncology, neuroscience and regenerative medicine with broad-ranging applications, such as disease model generation, the systemic study of disease initiating genes, selective genetic regulation and labeling, among others. CRISPR gene therapy is now in active development for treating cancers, blood disorders, cystic fibrosis, neurodegenerative diseases, and even for COVID-19. Outside of biomedicine, CRISPR has also emerged as an important tool to improve the traits of crops and in mosquito population control. This is a very exciting and fast-paced field and Abcam is working to be at the forefront to provide the best cell engineering solutions for researchers.
YK: HMEJ is different from the NHEJ as HMEJ has the homology arms (800 bp). In the case of the HMEJ method, we can knock-in the target (transgene) precisely and in the correct orientation.
YK: Previously, we have deleted up to several mega-base pairs (1,000,000bp).
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