As the 2020 Nobel Prizes have been awarded, it appears we will no longer have to wait to see CRISPR earn its rightful place in the upper echelons of science…
Originally discovered by Spanish scientist Dr. Francisco Mojica, the clustered regularly interspaced short palindromic repeats (CRISPR) was for a long time seen as a simple series of repetitive patterns in the DNA of single-celled prokaryotes such as archaea and bacteria. Even the simplest of organisms need a defense mechanism towards foreign invaders, such as bacteria-infecting viruses or bacteriophages. CRISPR is the basis of a fairly primitive immune system and works by “remembering” the DNA of viral invaders by chopping up and incorporating sequences within its CRISPR patterns. The CRISPR-associated protein 9 (Cas9) can recognize the DNA sequence stored within the bacterial CRISPR patterns and, upon any subsequent infection, cut DNA molecules with a matching sequence.
How convenient would it be if we could simply remove an aberrant piece of DNA or modify it to produce a desired protein? By providing it with the right template, CRISPR-Cas9 was mobilized into such a gene editing tool. CRISPR’s applications now range from in vivo gene therapy to ex vivo chimeric antigen receptor (CAR) T-cell engineering, including possibilities in non-medical fields such as agriculture with the potential to optimize plant growth to increase crop yields. Although CRISPR is not the first gene editing technology (transcription activator-like effector nucleases – TALEN and zinc-finger nucleases-ZFN have been around for quite some time), it has become the easiest and fastest to use. The scientists who collaborated on CRISPR’s functional development in the early 2010’s, Dr. Jennifer Doudna and Dr. Emanuelle Charpentier, were awarded the Nobel Prize in Chemistry earlier this month for their accomplishments.
But as Peter Parker has taught us “with great power comes great responsibility” and recent events have clouded CRISPR advancements. In 2018, Chinese scientist Dr. He Jiankui performed germline-genome editing on two in vitro fertilized (IVF) babies, modifying their expression of the chemokine receptor 5 (CCR5), ultimately hoping to decrease their future risk of contracting the human immunodeficiency virus(HIV). Such proceedings were met with a wave ethical concerns and complaints within the scientific community, pointing to the risk of off-target and unintended effects, as well as eugenics and designer-babies with above average athletic skills, high intelligence, and low risk of developing various diseases. Jiankui’s proceedings ultimately resulted in his arrest and imprisonment. Nevertheless, this reckless experiment should not tarnish the advances and ever-expanding potential of this field but instead should teach us to proceed with caution and regulation.
In honor of Synthego’s World CRISPR Day, we have decided to share with you one of many possible applications of CRISPR when it comes to hybridoma-based antibody research. Scientists in the Netherlands used CRISPR technology to switch the antibody isotype, as well as modify the final output of the hybridoma cells (now producing antibody fragments as opposed to full-length antibodies), all the while maintaining the targeted antigen specificity.
As the constant region plays a dire role when it comes to antibody function, having the right isotype is key when considering a finalized monoclonal antibody-based product. Such is the case with IgG1, which have an antibody dependent cellular cytotoxicity (ADCC) effector function (ex: Herceptin®, trastazumab, which targets and destroys HER2 overexpressing cancer cells with the help of immune cells) as well as IgG4 antibodies that lack effector functions but work great in blocking checkpoint inhibitors (ex: Keytruda®, pembrolizumab, which prevents the interaction of cancer cells with PD-1 expressing T-cells ultimately preventing their in-tumor suppression). Although the final antibodies discovered through hybridoma-technologies are often optimized via complementarity-determining region (CDR) grafting onto a set, and often human, antibody isotype construct, optimizing the desired antibody within the original hybridoma cell remains quite a feat.