Abstract
Laura J. Lambert
I walked into the laboratory in 2012 as a first-year graduate student on my third rotation. I was nervously excited to prove my worth and, in return, see if the lab was going to be the great fit I had hoped it would be. It was unusual to rotate in a university core facility, but learning to modify DNA in live animals was the reason I came to the University of Alabama at Birmingham (UAB).
My task was to establish the genetic modification of rats at UAB. I was handed protocols for producing TALENs and given a box of reagents referred to as “molecular Legos” to build them. As a cloning novice, the process was quite painful. Learning microinjection and surgical skills was simultaneously a joy and incredibly frustrating. We made the first genetically modified rats at UAB using a TALEN to the cystic fibrosis (cftr) gene in just under a year. Then, everything began to pick up speed as CRISPR began its revolution of our field (and many others).
It amuses me now to think how archaic this story sounds to somebody just starting out in the field. But 8 years feels more like 80 when one thinks of the astonishing progress in the field. We began our CRISPR endeavors by cloning guides into expression plasmids and co-injecting them alongside a Cas9-expressing plasmid. Trying to keep up with the ever-advancing protocols being published, we moved swiftly along to plasmid and polymerase chain reaction (PCR)-based in vitro transcription for guide and Cas9 RNA.
Around the time I finished my PhD in 2016 and assumed the role of Assistant Director of UAB's Transgenic and Genetically Engineered Models (TGEMs) Core Facility, we were largely using purified protein and two-part synthetic guide RNAs (sgRNAs). As a newly minted postdoctoral fellow, I began giving lectures and designing courses on the use of CRISPR and its ever-expanding repertoire. Before my eyes—and with my own hands—the production of genetically modified rats and mice began to speed up. Where I once quoted 18 months (if everything went smoothly) to produce gene knockout lines through embryonic stem cells, I was now quoting investigators 6 months or less for a simple knockout. Where I once cloned four or five guides into plasmids, laboriously produced RNA, and compared efficiencies to choose the best one, I was now using full-length sgRNAs delivered to the lab as easily as any PCR primer.
Now a faculty member and co-director alongside my long-time mentor, Robert Kesterson, I specialize in the creation of precision rodent models with patient-specific mutations. I use CRISPR to create the models and to try to correct the mutations through nanoparticle gene therapy. We work with UAB faculty, private foundations, and international institutes to train personnel in our workflows for CRISPR design or hands-on skills to produce rodent models. TGEMs recently became part of one of three national centers for precision animal modeling and has adapted to the influx in project demand by adopting new CRISPR protocols such as in vivo and ex vivo electroporation and use of alternative nucleases.
As large as our reach has become, I still feel the same rush of excitement when opening a genotype report as I did as a student when I turned on the ultraviolet light to see whether the PCR bands on my gels showed signs of CRISPR activity. What started as a graduate student project has become the center of my professional life. I often joke that I “grew up with CRISPR,” as we came into the field at the same time.
As I look back over my career, there is actually quite a lot of truth to that statement. We both entered with a lot of potential and were fortunate to have many talented scientists pour their knowledge into improving and shaping us. There is no way to overstate the impact CRISPR has had on my life, and what I hope will be a legacy of helping those affected by genetic disease as we search for answers.