Genetic Engineering Publications - GEG Tech top picks

In a review, researchers detail the development history of Principal Editing (PE), the latest evolution of CRISPR-Cas-based technologies. PE was proposed by a team of researchers in 2019, which is characterized by the absence of double-strand breaks (DSBs) or homology sequence patterns with variable application scenarios, including point mutations as well as insertions or deletions. The PE system consists of two parts: the master editors (PEs) and the master editing guide RNA (pegRNA). This PE system has developed and progressed rapidly over the last four years, with versatile advances in its architecture to increase editing efficiency, targeting and specificity, including a new pegRNA design, PE modification and improved delivery. Moreover, despite its relatively recent inception, PE has been widely applied to correct pathological mutations associated with genetic diseases, both in vitro and in vivo , presenting great potential for advancing the field of gene therapy from bench to bedside.

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. 

According to scientists at the Medical College of Georgia, a new edition of the CRISPR gene tool successfully suppressed the expression of Tspan2 gene involved in the differentiation of smooth muscle cells that help give strength and movement to organs and blood vessels. The researchers made a single-base change in the promoter region of Tspan2 and found that this inactivated the Tspan2 gene in the aorta and bladder without collateral damage unlike traditional CRISPR. This new editing only cuts a single strand of DNA unlike traditional editing which cuts double strands of DNA and makes unintended changes, which can be deadly to cells. Traditional CRISPR has 3 components: Cas9, molecular scissors that cuts both strands of DNA, guide RNA that directs the scissors to the right place and a repair model in case of problems. The new edition is composed of: Modified Cas9, named Cas9 nickase, which cuts a single strand of DNA, a complex called "main editor" with a reverse transcriptase and a PegRNA, the guide RNA of this new edition. One of the uses of this new CRISPR editing could be to create cell-specific knockout mice without extensive selection efforts that could also result in an exact model, for example, of the megacystis-microcolon-intestinal hypoperistalsis syndrome.

Three young patients with relapsing T-cell leukemia have now been treated with CAR T cells. Data from the clinical trial show how the donor CAR T cells were engineered using cutting-edge gene-editing technology to modify single letters of their DNA code so that they could fight leukemia. To generate "universal" anti-T CAR T cell banks for the study, the researchers used T cells from healthy donors. They then made modifications to the cells using basic editing. The steps were: the deletion of existing receptors so that a donor's T cells could be banked and used without pairing, making them "universal", the deletion of a "flag" called CD7 so that T cells would not kill each other, the deletion of a second "flag" called CD52 to make the edited cells invisible to some of the potent drugs administered to the patient during the treatment process, and the addition of a chimeric antigen receptor that recognizes the CD7 T-cell receptor on leukemic T-cells to combat T-cell leukemia. The clinical trial for this treatment is still open and aims to recruit up to 10 patients. The teams hope that the treatment can be offered earlier in the treatment pathway. With additional funding, they also hope to make it available for adults in the future. 

Jin-Soo Kim's team has discovered how to edit the mitochondrial genome to cure mitochondrial diseases. The first step was to adapt the basic editing system to induce C-to-T substitutions in mtDNA in mice because gene editing systems, such as CRISPR-Cas9, cannot edit mtDNA. Now the Kim lab has taken the next step: inducing A-to-G substitutions in mtDNA. Kim's lab has adapted basic editing systems to edit the mitochondrial genome in mice, inducing pathogenic mutations in the ND5 gene via the C-to-T substitution. They did this using DddAtox which acts on double-stranded DNA and was originally used in mitochondrial cytosine base editing systems. Using their own genome-wide library of DNA-binding transcription activator-like effector proteins (TALEs), they created a system to induce C-to-T modifications by fusing the TALEs to split halves of DddAtox and an uracil glycosylase inhibitor. In this system, the TALE acts in the same way as a CRISPR sgRNA by directing the DddAtox enzyme to a target sequence. Now, the ability to also make A-to-G substitutions allows the theoretical correction of about 83% of pathogenic single nucleotide mutations in the mitochondrial genome. The implications of the study also extend to mitochondrial genetic variations related to cancer and diabetes.