Genetic Engineering Publications - GEG Tech top picks

Genetic control in mammalian cells is essential for the development of safe and effective gene therapies. Current methods are associated with certain drawbacks, such as undesirable immunological responses, limited efficacy and overexpression of therapeutic genes. Current gene transfer technologies, such as adeno-associated viruses (AAVs), have difficulty achieving conditional and reversible gene control. Toxic ligands, leakage and high ligand concentrations, as well as small dynamic range are some of the limitations associated with current RNA-based systems. In a recent study researchers describe the pA regulatory system, inserted into cells by CRISPR-Cas9, based on a ribonucleic acid (RNA)-based switch to regulate mammalian gene expression by modulating the cleavage of a synthetic polyA signal (PAS) at a transgenic 5' untranslated region. (UTR). This technique differs from traditional riboswitch systems in that the PAS is present in the 5' UTR, combines the effects of numerous aptamers and uses two processes of Tc binding and alternative splicing. However, the new system can only use Tc as an inducing ligand, which cannot effectively penetrate all body tissues.

The methods present in this article decouple aspects of kinetic and thermodynamic properties of the Cas9–DNA interaction and broaden the toolkit for investigating off-target binding behavior.

Using CRISPR/Cas9 technology, researchers have devised a method to deliver a CAR gene to a specific locus, TRAC, in T cells. This targeted approach yielded therapeutic cells that were more potent even at low doses; in a mouse model of acute lymphoblastic leukemia, they outperformed CAR T cells created with a randomly integrating retroviral vector.

In this chapter, the authors discuss the current state of epigenomic profiling and how functional information can be indirectly inferred. They also present new approaches that promise definitive functional answers, which are collectively referred to as 'epigenome editing'. In particular, they explore CRISPR-based technologies for single-locus and multi-locus manipulation. Finally, they discuss which level of function can be achieved with each approach and introduce emerging strategies for high-throughput progression from profiles to function.

A modified version of Cas9 with a fusion of the hormone-binding domain of the estrogen receptor allows reversible control of Cas9 activity with high efficiency at multiple loci with 4-hydroxytamoxifen treatment.

In this work, the authors established neuronal models of FTLD-Tau by Neurogenin2-induced direct neuronal differentiation from FTLD-Tau patient iPSCs. They found that FTLD-Tau neurons, either with an intronic MAPT mutation or with an exonic mutation, developed accumulation and extracellular release of misfolded tau followed by neuronal death, which we confirmed by correction of the intronic mutation with CRISPR/Cas9. FTLD-Tau neurons showed dysregulation of the augmentation of Ca2+ transients evoked by electrical stimulation.

This FTLD-Tau model provides mechanistic insights into tauopathy pathogenesis and potential avenues for treatments.

The use of the Cas9/CRISPR system for efficient generation of precisely modified human pluripotent stem cells without secondary deleterious mutations in the untargeted allele
can be challenging. In this article, Howden and colleagues describe a variant of Cas9 that has been fused to a peptide derived from human Geminin to facilitate its degradation
during G1 phase of the cell cycle when DNA repair by NHEJ predominates. Using this variant (SpCas9-Gem) they demonstrate reliable and efficient derivation of both knockin
reporter iPSCs and genetically repaired patient-specific iPSC lines free of NHEJ-mediated indels at the target locus.

In the review, the authors provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.

On June 19, Emmanuelle Charpentier, Jennifer A. Doudna and Zhang Feng, were announced as the winners of the 2016 Tang Prize in Biopharmaceutical Science, which recognizes original biopharmaceutical or biomedical research that has led to significant advances towards preventing, diagnosing and/or treating major human diseases to improve human health. 

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 develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas9 delivered by a bacteriophage. They show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

Here the scientists conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, they collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. They find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. They further demonstrate that the best predictions can significantly reduce the time spent on guide screening.

There has been prolonged and significant interest in manipulating the genome for a wide range of applications in biomedical research and medicine. An existing challenge in realizing this potential has been the inability to precisely edit specific DNA sequences. In this review, the authors will discuss the impact of CRISPR/Cas9 on biomedical research and its potential implications in medicine.