Gene editing tools such as CRISPR are revolutionizing cell line development and engineering due to the speed and ease of performing edits. Here we explore the impacts that first generation and second generation tools are having and their future potential.
This extract is part of a whitepaper on the use of CRISPR/CAS9 and other gene editing tools in cell line development and engineering. Download the full whitepaper for free here.
First generation genome-editing tools
Several recombinases have been used for targeted site approaches, among which the Cre/Lox and the Flp/FRT were mostly utilized. Moreover, recombination-mediated cassette exchange (RMCE) technology is attracting interest by the industry for targeted gene insertion. This technology had good achievement in increasing the success rate and reducing timelines for the generation of stable industrial-grade CHO cell lines expressing monoclonal antibodies reliably.
Second generation genome-editing tools
This group includes different endonucleases like meganucleases, zinc finger nucleases (ZFN), CRISPR/Cas9, and transcription activator-like effectors nucleases (TALEN). Both ZFN and TALEN technologies rely on the ability to customize a DNA-binding domain for a specific sequence (the targeted sequence for cleavage) combined to a nuclease effector domain. The CRISPR/Cas9 technology has already been validated in CHO cells and reduces the production variability between clones. ZFN, TALEN and CRISPR/Cas9 technologies are however mostly used for the gene-specific knockout.
One of the critical challenges is still to identify good hotspots in the host genome that will allow good expression levels and stability. Such specific integration may not be a one-size-fits-all approach as some therapeutic proteins may require a particular level of expression to fold correctly or to acquire adequate quality attributes (e.g., glycosylation, proteolytic processing) Eventually, the discovery of further naturally occurring RNA-guided nucleases offers added targeting flexibility. Researchers have also engineered Cas9 enzymes to exhibit relaxed PAM specificities. These crucial advances broaden the number of target loci responsive to RNA-guided genome editing. Other scientists have engineered Cas9 nuclease and managed to increase its DNA specificity dramatically.
Besides, “base editing” is a reality, a strategy to generate point mutations in the genome thanks to the fusion of dCas9 to a cytidine deaminase enzyme that operates on ssDNA.
Remarkably, CHO cells, where simultaneous (‘multiplex’) disruption of FUT8, BAX and BAK genes by CRISP/ Cas9 succeeded and already showed increased resistance to apoptosis. Furthermore, the ZFN knockout approach accomplished the specific deletion of the GS and the DHFR genes in CHO cells, thus improving the selection stringency of the generated cell lines. Finally, both ZFN and a CRISPR/Cas9 approaches were also used for FUT8 gene specific knockout. As a result, these cell lines completely abolished fucosylation on the Fc domain of IgG.
An additional, though less frequently used tool, is the mammalian artificial chromosome expression (ACE) technology. Such minigenome works as an autonomous genetic element that multiplies with the cells. Various regulating elements contained within its DNA sequence make that vector customizable for an optimized expression.
Also, the PiggyBacTM (System Bioscience inc.) transposon system uses an efficient transposase purified from the cabbage looper (Trichoplusia ni) to integrate the gene of interest into the host genome easily. It has shown improved yields for stable production of antibodies in CHO cell lines.
What is being achieved with genome-editing? What will be obtained soon?
Introduction of additional N-glycan target sites into desired positions on the protein backbone by genetic mutation has been used to create glycoproteins with enhanced levels of glycosylation (overexpression of sialyltransferases and other glycosyltransferases, inhibition of sialidases) and consequently sialylation, leading to extended serum half-life and improved in vivo activity. (N-glycans can also be crucial for protein folding).
Thanks to a comprehensive Zinc-finger nuclease (ZFN) knockout screen of glycosyltransferase genes and the identification of key genes that control decisive steps in N-glycosylation in CHO, it is possible today to provide homogeneous glycoforms.
The overexpression of Bcl-2 or Bcl-xL has shown precise and efficient inhibition of apoptosis in rCHO cell cultures by enhancing the culture longevity, cell viability, and endurance to environmental stresses. Consequently, rendering greater yields of therapeutic protein, which is a definite economic advantage in the biopharmaceutical industry. Similarly, down-regulation of caspases, such as caspase-8 and -9, and knockouts of pro-apoptotic genes, such as Bax and Bak, enhances the viability of both batch and fed-batch cultures. (Down-regulation of such genes can be done with various genome-editing techniques like ZFNs, TALENs or CRISPR-associated (Cas) system).
Therapeutic protein expression regulation by smart promoters and epigenomic reprogramming.
Shortly research labs will be able: to design transcriptional control systems, synthetic promoters, controlling the timing of gene expression at will by trigger-inducible transcription factors (TF), protectin against chromatin silencing. In addition to changing the DNA sequence of a given genome, researchers have already begun to edit the epigenome in order to alter the regulation of a target gene. Moreover, the use of effector fusions can significantly expand the repertoire of genome engineering modalities achievable using Cas9. Furthermore, non-editing CRISPR tools are helping in screening engineered cells for a phenotype of interest with speed and precision.