Genetic Engineering Publications - GEG Tech top picks

CRISPR genome editing has served as a powerful tool to delete or modify DNA sequences and study the resulting effect. Now, researchers at the Gladstone Institutes and UC San Francisco (UCSF) have co-opted the CRISPR-Cas9 system to forcibly turn on genes rather than edit them in human immune cells. The method, known as CRISPRa, allowed them to discover genes that play a role in immune cell biology more thoroughly and quickly than before. The study, published in the journal Science, is the first to successfully use CRISPRa on a large scale in primary human cells, which are cells isolated directly from a person. In the new work, Marson, Steinhart and co-first author Ralf Schmidt, MD, worked with their colleagues to adapt CRISPRa and CRISPRi to work at high efficiency in primary T cells, something never done before. Improving the efficiency of delivery of the CRISPRa or CRISPRi machinery into cells was essential to enable genome-wide experiments and accelerate discovery. Marson's lab is currently studying some of the individual genes identified in their screen, and working to further leverage CRISPRa and CRISPRi to discover genes that control other critical traits in human immune cells.  

Scientists in Japan have used CRISPR-Cas9 technology to stop human immunodeficiency virus type 1 (HIV-1) replication in latently infected T cells that can’t be controlled using existing drug treatments. The gene-editing approach effectively disrupts two regulatory HIV-1 genes, tat and rev, which are essential for viral replication. Describing their in vitro studies in Scientific Reports, the researchers at Kobe University Graduate School of Medicine and Kobe University Graduate School of Health Sciences say initial results indicate that using CRISPR-Cas9 to target HIV-1 regulatory genes may offer a new approach to achieving “functional cures.”

The race to the clinic is reviving for a ready-made alternative to autologous CAR-T cell therapy, even as concerns about chromosomal abnormalities persist. The Advanced Regenerative Medicine Therapy designation, which makes the therapy eligible for accelerated approval, will also help remove a veil that has hung over standard CAR-T cell therapies since October, when the FDA put all trials of competitor Allogene Therapeutics on hold following the detection of a chromosomal abnormality in a patient who received ALLO-501A in a Phase 2 trial. The FDA's green light for CRISPR Therapeutics dispels broader concerns that the agency views this type of genotoxic safety event as an intractable problem for the entire class of allogeneic CAR-T therapies. Today, many companies are eliminating loci associated with the MHC-I to avoid host T cell recognition of transplanted CAR-T cells. Companies also equip their T cells with a variety of safety switches and performance enhancers.

However, as the complexity of the assembly increases, the risk of off-target effects also increases. This may be important from a safety perspective, given that most cancers lack unique antigens. Achieving rapid remission and re-dosing if necessary, can minimize the toxic effects that CAR-T cells can have on healthy tissues expressing the targeted antigen.

In this work, the authors  assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) for CRISPR system to knock out endogenous TCR expression. Using nuclease-expressing plasmid DNA, they achieved up to 19.9% and 12.2% knockout of TCR expression in primary T cells with CRISPR/Cas9 and TALENs, respectively. In contrast, delivery of TALEN mRNA by electroporation resulted in high viability and TCR knockout efficiencies of up to 78.8% for the TCR α chain and 81.2% for the β chain on day 6 after electroporation.