Gene Targeting Service Verified with Industry-leading Quality Control Measures
At Biocytogen, we take pride in our quality control measures when we target and edit genes for our mouse models and drug development endeavors. From preventing potentially disastrous results before they occur to instituting reliable, tried-and-true protocols, proper attention to quality control when gene-editing can save clients both time and resources. For example, based on our internal data, the random insertion rate of gene-edited mice generated by CRISPR based EGE method is about 32%, and almost half of them can’t be segregated from the properly targeted allele through breeding.
Quality control, we believe, is integral and vitally important not only in matters of quality assurance during the model production steps but also in the end-user application process. The standard procedures involved in the QCs may seem costly, labor-intensive and time-consuming, but it is absolutely rewarding from a long-term perspective. To discuss some aspects of quality control in gene editing, we would like to introduce you to two expertise, in-house scientists at Biocytogen: Min Deng, Ph.D. and Dan Wang, Ph.D.
Who are you?
Min Deng, Ph.D. (MD) was a research assistant professor and manager at the University of Rochester, specializing in neuroscience, development, transgenic and knockout technologies. At Biocytogen, Min is a senior scientist II and a senior project manager. She has more than 18 years of expertise in the production and application of gene-edited cell lines and rodent (mouse and rat) models. She is keen on anything related to biology. She likes music, travel and good food.
Dan Wang, Ph.D. (DW) graduated from Cornell University and obtained postdoctoral training at Harvard Medical School, specializing in diabetic embryopathy, stem cell, and regenerative medicine. Upon joining Biocytogen, Dan has been building up the company’s expertise in genome editing and program management. Dan is a senior scientist providing targeting strategy designs and a project manager leveraging her technical expertise, scientific conceptual understanding, and management capacity to provide strategic support with total solutions to accommodate the researchers’ request. Dan thrives working in a dynamic environment by building multicultural teams as well as driving internal alignment and external collaborations.
Can you describe a time where you experienced a troublesome situation that could have been prevented by better Quality Control?
MD: I used to be a manager in the transgenic core at University of Rochester. When we first attempted to generate a floxed mouse model using CRISPR/Cas9 method, we didn’t test sgRNA activity in vitro. Instead of targeting vectors, we used DNA oligo as the DNA template. After performing several rounds of microinjection, even though we screened many gene-edited F0 pups, they all had indels.
DW: Yes, we have experienced several challenging situations since the first utilization of our CRISPR/Cas9-based EGE™ platform in 2014. Due to the lack of a proficient QC system at the time, several models generated in the first batch were problematic. Undesired phenotypes were observed, which theoretically wouldn’t be resulted from the designed gene manipulation itself, suggesting that undesired cuts or random integrations might have occurred. In response, we have reinforced and optimized the system since then by adding three important QC steps to correct the situation. We are quite confident that we can now deliver the models with premium quality for our collaborators.
What are the Quality Control systems in place at Biocytogen?
Biocytogen offers three important quality control systems when the CRISPR/Cas9-based EGE™ technology is applied:
- Southern blot analysis is performed during F1 mice screening to exclude the possibility of random integration – we only deliver F1 mice with a clean genetic background without random insertion issues. Our data showed that when the ES cell method is used for the preparation, the probability of random insertion is about 20%; if CRISPR/Cas9 is used, the probability of random insertion increases to 32%. This QC step is vitally important;
- Two rounds of PCR and sequencing analysis are performed on F0 founder stage to screen out undesired genome edits; two rounds of PCR and sequencing analysis are performed on F1 generation to ensure the whole targeted region are correct, excluding the ones with undesired genome edits and keeping the ones with desired edits only;
- Highly predicted off-target site screening is performed for F1 mice. For each targeting site, we design at least eight sgRNAs and test their activity in vitro using our UCA kit to determine one appropriate sgRNA with high specificity and high activity, which dramatically decreases off-target effects.
What makes Quality Control at Biocytogen stand out?
MD: For our improved CRISPR/Cas9 based EGE system, instead of an oligo DNA template, we co-inject the targeting vector DNA with Cas9 mRNA and sgRNA. The targeting vector includes 5’ and 3’ homologous arms (~ 1.5 kb) which reduce random insertion and off-target effects tremendously.
DW: There are many things that make us stand out. If you want me to pick one, I would say the Southern blot analysis for F1 generation of mice generated by CRISPR/Cas9-based EGE™ technology. So far Southern blot analysis is the most effective way to exclude the F1 mice with random integration issues. Unfortunately, most other vendors either haven’t realized that or could not afford to perform Southern blot analysis, which requires a substantial amount of know-how to master.
Indeed, research has demonstrated that even in on-target mutagenesis cases, DNA breaks caused by CRISPR/Cas9 may result in large-scale deletions or other undesired results (Kosicki). Improvement in confirming on-target alleles of interest is still needed (Ayabe). Utilizing the lessons learned and wisdom gained from over a thousand completed past projects, our scientists are focused on providing our clients with the most up-to-date, innovative protocols, technologies, and strategies.
Biocytogen offers gene-editing services based on multiple modalities, including CRISPR/Cas9 Extreme Genome Editing (EGE™), ESC-based gene editing, and transgenic technology, all backed with our industry-leading quality control as a top priority. With a team of over 200 scientists in China in addition to those in Boston, MA USA, Biocytogen is a global, multi-cultural company that is well versed in collaboration and communication. You can find Biocytogen’s work in numerous publications in high profile journals such as Nature, J. of Immunology, and Oncogene.
Kosicki, M., Tomberg, K., & Bradley, A. (2018). Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nature biotechnology, 36(8), 765-771.
Ayabe, S., Nakashima, K., & Yoshiki, A. (2019). Off-and on-target effects of genome editing in mouse embryos. Journal of Reproduction and Development, 65(1), 1-5.
Updated March 16 2020.