Researchers at Sanofi report that cas-CLOVER (deactivated Cas9 fused to the C-terminus of Clostridium Clo051) gene editing has the potential to notably enhance recombinant protein production in Chinese hamster ovary (CHO) cells, potentially offering an alternative to CRISPR-Cas9 editing.
“We were looking for a gene editing technology with freedom to operate that yielded the same benefits of a CRISPR-Cas9 system, such as ease and efficiency. Cas-CLOVER allowed this with the chance of less off-target effects,” Tiffany McLamarrah, PhD, principal scientist at Sanofi, tells GEN. The cas-CLOVER editing system has already shown an approximate 90% editing efficiency in HEK-293 cells. Now it is beginning to be studied in CHO cells.
A team led by McLamarrah, who is the first author of a recent paper, evaluated the efficiency of cas-CLOVER to knockout the glutamine synthetase locus in CHO cells. More than 100 clones were generated from 30 different serum-free-suspension adapted CHO-K1 host cell lines. In 98 of those clones, complete knockout occurred. That phase of the project took only six weeks, which is comparable to CRISPR-Cas9 knockouts.
Next, glutamine synthetase knockout clones were chosen randomly as production vehicles for monoclonal antibodies. McLamarrah and colleagues reported that titer increased “up to 14.4-fold, compared to their wild-type parental hosts.” That, she says, merely “highlights the differences among the CHO host cells and the vector system used to generate the pools.”
To achieve increased productivity, reducing glutamine synthetase expression alone wasn’t enough. The researchers also had to attenuate the exogenously expressed glutamine synthetase to achieve a higher titer of knockouts when compared to wild-type cells.
Large indels ease KOs/KIs
Indels resulting from cas-CLOVER editing are larger than those typically achieved by CRISPR-Cas9 editing. For example, cas-CLOVER gene editing resulted in indels ranging from a gain of 13 base pairs (bp) to a loss of 128 base pairs. “The most common deletions were -12 bp and -26 bp at a 3.7% and 3.3% frequency, respectively,” they note. Furthermore, “92.7% of indels started within the 73 bp range comprised of the two sgRNAs and their respective protospacer adjacent motifs.” Cleavage in that range is considered probable.
“The staggered cuts generated by cas-CLOVER may lead to these larger indels that may allow for more efficient knock-in, and also may lead to easier generation of knock-outs,” McLamarrah elaborates. “Deleting 20–30 bps allows us to eliminate specific amino acids necessary for the function of certain genes, not always requiring a frameshift, and ensuring a complete knock-out of the protein with less potential for future rearrangements yielding a functional protein again.”
High resolution next gen sequencing (NGS) and fragment analysis capillary electrophoresis (FA-CE) indicated that 97% of the indel sizes were within the 1 bp margin of error.
In addition to NGS and FA-CE analysis, gene knockout was also confirmed behaviorally. When the GSKO clones were inoculated in media lacking glutamine supplements, they exhibited glutamine auxotrophic behavior. Viability declined to about 70% by day four, and 30 to 40% by day six. Nearly all the cells that were supplemented with glutamine, however, remained viable throughout the nine days of the study.
The project showed that productivity depends heavily on the CHO-K1 host cell lines that are chosen. Therefore, comprehensive host cell line screening is important. Next, McLamarrah says, “We need to determine the off-target effects in our CHO cells and are working on more knock-ins.”