Genetic Engineering in the Press by GEG

Probiotics, widely regarded as a treasure trove in the field of microbiology, are currently finding new applications in medicine, animal care and the food industry. However, it is often difficult to use probiotics in their existing form, due to the varying effects of different strains on the health of different individuals. Consequently, it is also difficult to find concrete evidence to support the proposed benefits of probiotics. Fortunately, these challenges can be overcome thanks to the considerable advances we've seen in genetic engineering over the past decade, particularly following the introduction of the hugely popular CRISPR-Cas editing system. By modifying, deleting or introducing specific genes with these tools, we can tailor the activities of probiotic organisms to our health needs. To support the researchers interested in pushing the limits of the field, Professor Nan Peng from the Huazhong Agricultural University, China, along with his colleagues, recently published a review article in Volume 5 of BioDesign Research summarizing the latest advances and hurdles related to the engineering of probiotics with CRISPR-Cas.

The incidence of Parkinson's disease (PD) increases with age and is the second most common neurological disease. PD is caused by the degeneration of dopaminergic neurons in the substantia nigra, which produce dopamine (DA). In particular, astrocytes are essential to the brain's immunological response in Parkinson's disease, as demonstrated by animal models and post-mortem investigations. Tyrosine hydroxylase (Th) is an enzyme present in astrocytes that is crucial for DA production. The optimal approach to managing PD involves the use of levodopa (L-DOPA), a precursor that facilitates DA formation and substitution. This treatment has a limited duration of efficacy, generally extending over five years. In a recent study published in Gene Therapy , researchers use the CRISPR-Cas9 system to induce dopamine (DA) synthesis in the brain of a rat model of Parkinson's disease. The researchers analyzed the rat genome to identify potential guide ribonucleic acid (sgRNA) sequences that were highly specific and did not align with other areas of the rat genome. This led to the identification of 13 sgRNAs for gene activation. Among the 13 th sgRNAs identified in the analysis, the researchers discussed the results of the TH4 sgRNA, as it achieved the highest levels of Th protein expression.

Depleted T cells exhibit poor responses to tumor antigens, and limited proliferation and persistence in vivo. With high expression of inhibitory receptors and low levels of effector proteins, they are generally associated with poor clinical outcomes. A team of researchers set out to understand the origin of this depletion using high-throughput genomic technologies, in the hope of improving the outcomes of cellular immunotherapy for patients. To identify the molecular and genetic determinants of T cell exhaustion in vitro , the researchers developed a chronic TCR evolutionary stimulation assay that summarized the state of T cell exhaustion. They then subjected depleted T cells to genome-wide CRISPR-Cas9 screens, with surprising results. After an intensive search, the team found such a target: the AT rich interaction domain 1A, otherwise known as Arid1A. This gene is involved in the regulation of transcription through chromatin remodeling in the activating and promoting regions of genes. CRISPR-Cas9 inactivation of Arid1A resulted in marked improvements in T cell performance in vitro and in vivo.

There are different types of CRISPR system, and the one on which researchers have focused to describe the three-dimensional structure in detail is called CRISPR-Cas13bt3. What's unique about it is that it's very small. Usually, these types of molecules contain around 1,200 amino acids, whereas this one only contains around 700. Smaller size is a plus, as it allows better access and delivery to target editing sites. To obtain the three-dimensional structure, the researchers used a cryoelectron microscope to map the structure of the CRISPR system, placing the molecule on a thin layer of ice and projecting an electron beam through it to generate data which was then processed into a detailed three-dimensional model. CRISPR-Cas13bt3 is totally different from CRISPR-Cas9: the scissors are already there, but they have to hook onto the RNA strand at the right target site. To do this, it uses a binding element on those two unique loops that link the different parts of the protein together. The researchers then used their findings to refine the tool to increase its precision, and tested its activity and specificity in living cells. They found that in cell cultures, these systems were able to target much more easily.

The rare and fatal genetic disease CD3 delta severe combined immunodeficiency, also known as CD3 delta SCID, is caused by a mutation in the CD3D gene, which prevents the production of the CD3 delta protein needed for normal T cell development from blood stem cells. Currently, bone marrow transplantation is the only treatment available, but the procedure carries significant risks. In a study published in Cell, researchers showed that a new genome editing technique called base editing can correct the mutation that causes CD3 delta SCID in blood stem cells and restore their ability to produce T cells. The basic editor corrected an average of nearly 71 percent of the patient's stem cells in three experiments. The researchers then tested whether the corrected cells could give rise to T cells. When the corrected blood stem cells were introduced into artificial thymic organoids, they produced fully functional and mature T cells. The corrected cells remained four months after transplantation, indicating that the basic editing had corrected the mutation in the true self-renewing blood stem cells. The results suggest that the corrected blood stem cells could persist over the long term and produce the T cells that patients would need to lead healthy lives. 

Beta thalassaemia and sickle cell disease are caused by genetic mutations that affect the adult form of haemoglobin. However, some people have few or no symptoms despite having one of the mutations that normally cause these disorders, because their bodies continue to produce foetal haemoglobin into adulthood. A CRISPR-based treatment, called exa-cel, has therefore been developed that aims to mimic this by extracting blood-producing stem cells from people's bone marrow, using CRISPR gene editing to deactivate the gene that disables fetal haemoglobin production, and then reintroducing the edited cells into each person. Initial promising results for the first people to be treated have recently been published. Results from phase II trials testing the effectiveness of the approach in 75 people have also been announced. In addition, clinical trials are being expanded to include children under 12 years of age after the therapies proved effective in ongoing trials involving people aged 12 to 35. The aim is to treat children early enough to prevent them from developing lasting damage from these inherited disorders.