Genetic Engineering Publications - GEG Tech top picks

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.

Here, the authors characterize the effect of different culture temperatures on CRISPR-Cas9 mediated genome editing and find that the genome editing efficiency of CRISPR-Cas9 is significantly hampered by hypothermia treatment, unlike ZFN and TALEN. In addition, hyperthermia culture condition enhances genome editing by CRISPR-Cas9 in some cell lines, due to the higher enzyme activity and sgRNA expression level at higher temperature. This study has implications on CRISPR-Cas9 applications in a broad spectrum of species, many of which do not live at 37 °C.

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. 

This review introduces the clinical manifestations of three distinct neurodegenerative diseases and the applications of the gene-editing technology on these debilitating diseases.

Herein, the authors summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins (RNPs) for genome editing. Additionally, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

In this review, the authors will discuss the specific applications of gene-editing technologies in human HSPC, as informed by prior experience with gene addition strategies. HSPC are desirable but challenging targets; the specific mechanisms these cells evolved to protect themselves from DNA damage render them potentially more susceptible to oncogenesis, especially given their ability to self-renew and their long-term proliferative potential. They further review scientists’ experience with gene-editing technologies to date, focusing on strategies to move these techniques towards implementation in safe and effective clinical trials.

In this work, the scientists compre TALEN and CRISPR to target two loci within the EGFP gene. They find that the CRISPR system induced targeted genomic deletion more efficiently and precisely than TALENs. However, TALENs stimulated homology directed repair more efficiently than CRISPR system and caused fewer targeted genomic deletions.

These data suggest that the choice of genome editing tool should be determined by the desired genome editing outcome. Such a rational approach is likely to benefit research outputs for groups working in fields as diverse as modification of cell lines, to animal models for disease studies, or gene therapy strategies.

In this work, the scientists show that using phosphorothioate-modified oligonucleotides strongly enhances genomeediting efficiency of single-stranded oligonucleotide donors in cultured cells. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular.

Over the last years, new genetic engineering technologies have emerged and undoubtedly show up game-changing. Genome engineering is since a long time

Genome editing of human ES/iPS cells has been limited by technical difficulties that result in a low efficiency of homologous recombination (HR) in human ES/iPS cells.

In this work, the authors demonstrated that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Their findings would facilitate genome editing study using human ES/iPS cells.

Here the authors review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR–Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field.

Here the scientists report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly (‘Gibson assembly’) that is labour- and cost-effective, accessible, rapid and scalable.

Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, Jin-Soo Kim review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)–Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs). He specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR–Cas9 genome editing approaches that has revolutionized the field.