Fenglin Wang, Director of Cell Line Development and Protein Sciences, began the podcast by providing background on CRISPR/Cas9 as a molecular biology gene editing tool. She explained that the CRISPR/Cas9 gene editing system is adapted from a natural prokaryotic defense mechanism to bacteriophage. To simplify, the CRISPR/Cas9 system cleaves the phage DNA once it has been incorporated into the bacterial genome to keep the phage from reproducing. CRISPR is an acronym that stands for “Clusters of Regularly Interspaced Short Palindromic Repeats,” and Cas9 is the most well-researched variant of the class of Cas nucleases used within this gene editing function. The research community has adapted this mechanism to revolutionize how genetic modifications are performed in prokaryotic and eukaryotic cells.
Next, I asked Zane Starkewolfe, Director of Corporate Development, about the origin of CRISPR/Cas. He shared that research can be found on CRISPR that dates back to the late 1980s and other work was conducted throughout the first decade of the 2000s. However, in 2012, two pivotal research papers were published in the journals Science, one by Jennifer Doudna of UC Berkeley and Emmanuelle Charpentier of the University of Vienna and then another in PNAS by Giedrius Gasiunas of the Virginijus Šikšnys lab at Vilnius University. All these papers demonstrated the use of bacterial CRISPR/Cas9 as a simple, programmable gene-editing tool. He went on to say that in less than a year, the labs of Dr. Feng Zhang of the Broad Institute at MIT, and Dr. George Church’s lab at Harvard, reported success in adapting CRISPR/Cas9 for genome editing in eukaryotic cells in both mouse and human cells.
I then asked Zane why was there so much excitement about the use of CRISPR as a molecular biology tool. He described how the remarkable functionality of CRISPR is that it allows scientists to target specific locations within the genetic code of an organism, to cut out or replace a segment of DNA. Due to the high specificity and exactness of the utility, the applications have far-reaching potential. Thus, it has already become a molecular toolbox game changer in many fields of life science because it enables efficient, cost-effective, precision gene editing that has wide utility for development of biological therapeutics including cell and gene therapy, disease modeling, diagnostics, agriculture, industrial biology and more.
Then we discussed what makes CRISPR/Cas better than other gene editing systems. Zane said that many of the other gene editing systems utilized today such as Zinc Finger Nucleases, TALENS, the use of meganucleases or viral vectors like AAV, when compared with CRISPR/Cas9 are in the end very complex and time-intensive. They often require many more steps and are more costly. Also, CRISPR/Cas9 has a low “off-target” effects profile, which again makes it an ideal gene editing tool.
I asked Zane to describe some of the recent advancements using CRISPR technologies. He said that the advances are extensive and ongoing. One example he shared was CRISPRa and CRISPRi1, which are techniques to up and down regulate respectfully gene expression using “dead Cas9.” Dead Cas9 removes the nuclease capability of Cas9 but still allows for targeted binding to a double-stranded DNA sequence of interest using the highly specific single-guide RNA that is one of the cornerstones of CRISPR genome editing.
Fenglin added that another application is using CRISPR for Homology Directed Repair or HDR. This technique, in simple terms, repairs double-stranded DNA breaks. This is important for genome stability. The CRISPR-mediated HDR can be used to repair a break and also can create the break and then replace it with a small mutation or other larger sequences. These techniques have substantially opened the ability of researchers to make genome edits quickly and efficiently.
Next, I asked about the scalability of CRISPR. Zane explained that researchers across the globe are using CRISPR in high-throughput scenarios using libraries of single-guide RNA to discover new drug targets and develop rapid diagnostics, including using it in the fight against COVID-19, and an array of other applications.
I asked Fenglin about the applications of CRISPR in biopharmaceutical development. She talked about the many ways that CRISPR can impact the success of molecules by modifying or deleting sequences within the product genes themselves to optimize the product for its intended therapeutic purpose. For viral vaccines in particular, CRISPR can be used to edit the genes encoding the protein coat or membrane of the virus to deliver a greater immune response or reduce side effects in vivo. In addition, as Zane mentioned, up and down regulation of genes involved in the expression of proteins or involved in post translational modifications of the protein product are possible as well.
I followed up by asking how one might start making those improvements. She said that to do that we simply need to understand where things can be improved. Most scientists today have the advantage, in contrast to years past, of knowing that CRISPR is in their molecular toolbox to make those changes quickly and easily. Thus, those changes can be evaluated at a much faster pace than ever before.
Next, I asked Zane about other Cas constructs. He clarified that Cas9 is currently the most widely known nuclease in CRISPR experiments, and more specifically the Cas9 variant isolated from the bacterium Streptococcus pyogenes (SpCas9). Although Cas9 is the most widely used for genome editing, it does have certain limitations; it is not 100% efficient, can have off-target effects and it is relatively large, making it difficult at times to deliver into cells using common vectors.
I asked if there was a way around this. He said that to overcome the limitations with Cas9, several approaches have been taken by researchers, including identifying other naturally-occurring Cas enzymes such as Cas 12 or 14, but also performing genetic engineering directly on the Cas9 to produce a better, safer nuclease variant for genome editing. Fenglin clarified that the comparison Zane was referring to is for larger gene editing tools. Site directed single nucleotide mutagenesis still has good utility and compares favorably to CRISPR/Cas9 for very small edits, but it is rather impractical from a time, resources and cost perspective for larger edits. It cannot do all the varied things that CRISPR/Cas9 can accomplish, especially when compared to its ease of use and costs.
I pointed out that there are some really great websites that detail exactly how CRISPR/Ca9 functions at the molecular level and other sites that provide an overview of the scientific and legal history. Those links can be found at the end of this article.
We then discussed, given this complex background, WuXi Biologics’ approach to using CRISPR technologies. Fenglin described that historically; they have provided discovery and CDMO services to companies developing biological therapeutics. They specialize in providing technology platforms for cell line engineering and biomanufacturing. Thus, they are interested in applying CRISPR in those arenas.
I asked about some of the applications of CRISPR/Cas9 in biologics development. She explained that from their perspective, which represents the interests of their clients as well, they are looking to optimize or address situations surrounding three main areas. The first being to improve cell growth characteristics which can greatly impact the eventual manufacturing of the biologic; the second is to improve cell productivity and product titer which also has significant ramifications on manufacturing cost of goods; and the third, looking at ways to optimize or improve product quality.
I followed up by asking if she could go into detail about how CRISPR/Cas9 be used to help improve product quality. She explained using antibodies as an example. Antibodies are very complex molecules and sometimes certain individual monoclonal and bispecific antibodies can demonstrate high aggregation. They could also breakdown in solution or in vivo thus causing adverse immune responses or a drop in potency. In addition, there will be glycosylation profiles that cause adverse immune response events or cause the biotherapeutic to be cleared from the body easily. All of these scenarios are undesirable and CRISPR/Cas9 can help fix those types of issues.
I then asked Zane how CRISPR could be used at the cellular level to improve biologics manufacturing. He said that there are multiple ways CRISPR/Cas9 can be used to attack cell culture issues and genetically engineer production host cells by knocking out genes or knocking down or inducing gene expression. Editing the genome to improve on an already well-established cell line system is advantageous and cost effective, since these lines are well-characterized, most often have acceptable and proven safety profiles, and have demonstrated many times over to be effective producers. With the advent of CRISPR/Cas9 for gene editing, it has become easier and quicker than ever before to manipulate the genome of production host cells to improve their biopharmaceutical production.
Fenglin added that the end goal for cell culture is to improve or modify host cell robustness, growth rates, stability and longevity. Certainly, changes in culture media and other physical culture parameters can have positive effects on these as well, but there are limitations with each production host cell line that can only be overcome through cell engineering and this is where CRISPR can play a big role.
I asked her for some specific examples and she shared several. One example provided was optimization of parameters like regulating cellular apoptosis to prevent premature cell death. Another example was focusing on metabolic engineering genes to control the cell cycle to achieve faster cell growth or productivity. She went on to provide an example from a recent research publication where CRISPR/Cas9 was utilized by researchers to knockout specific amino acid catabolism genes in CHO cells to reduce secretion of growth inhibiting metabolic by-products like lactate and ammonium.2
Another research publication example of using CRISPR/Cas9 to induce protein expression and increase product titer was when researchers using elements of the Cre-lox system from the bacteriophage P1 and Flp/FRT system were used in eukaryotic cells to establish site-specific target gene insertion into high expression chromosomal loci.3
Also, as mentioned previously, using CRISPR/Cas9 to obtain the ideal glycosylation pattern for a recombinant protein4 is a common function and at WuXi Biologics, we have significant experience engineering cell lines using CRISPR/Cas9 to modify the protein glycosylation profile.
Zane added that some researchers are evaluating how CRISPR/Cas9 could impact the use of Chaperones5 and foldases to optimize protein assembly and folding which in turn may help to improve cell productivity and product titer. He stressed that for all of these examples, it is important to determine empirically if these genes can make an impact.
Next I asked about CRISPR being used in clinical trials. Zane explained that it is currently being used in clinical trials similar to how CAR-T or other cellular or viral gene therapy approaches are being used. Even though there have been some early successes, there are many challenges to using CRISPR in vivo because CRISPR/Cas9 is considered “foreign” and thus immunogenicity and fast clearance are huge concerns. Another area of concern is the degradation of the complex in cells before it has an opportunity to act. One fascinating approach that is being evaluated is tethering the CRISPR/Cas complex to a monoclonal antibody for site-directed delivery to a specific cell type such as a tumor or other specific tissues.6
Fenglin added that what Zane said was interesting because creating CRISPR/Cas-based therapeutics is an intriguing CMC and GMP manufacturing challenge from a supply chain perspective. There is the production of the Cas protein and the guide RNAs, which must be done GMP, but in the example Zane gave, you will also need to manufacture the mAb and the linker and then conjugation of all of the various components. Thus, all of these elements add up to a very challenging CMC program and supply chain.
Most companies will not have the expertise to conduct all of these activities. So you will have to outsource many of these activities and finding the requisite expertise will be difficult. In addition, trying to coordinate this amongst all the different vendors and CDMOs is a huge challenge. This is why WuXi Biologics have assembled all of these capabilities into their organization. WuXi Biologics along with WuXi STA, a WuXi AppTec company, can produce the mAb, Cas protein, chemical linkers and guide RNA and perform the conjugation all with facilities that are only a few hours from each other. Using their well-established project management program, they can be a single source for any company hoping to develop these novel CRISPR-based therapeutics.
I closed the interview by asking how companies hoping to use or make improvements to their cell lines and biologics could work with WuXi Biologics to determine if CRISPR/Cas9 is a feasible solution. Zane said that at WuXi Biologics, they recently took a research license from the Broad Institute on CRISPR-Cas9. Now they can offer it as a service for customers who are interested in using it for cell line engineering.
Fenglin expanded by saying interested companies should contact WuXi Biologics and describe the end goal. Then, Fenglin along with WuXi Biologics’ team of experienced cell line engineering scientists and cell biologists will work collaboratively with you to help determine if the use of CRISPR/Cas9 is needed. If so, they will conduct the work for you and together review the results and determination of next steps.
Technical/resource information on CRISPR/Cas gene editing
CRISPR/Cas Nobel Prize Announcement
CRISPR/Cas Legal / Patent Information