Genetic Engineering Publications - GEG Tech top picks

A new tool could reduce the cost of diagnosing infectious diseases. Researchers have developed a new, less expensive means of detecting nuclease digestion, one of the critical steps in many nucleic acid detection applications, such as those used to identify COVID-19 and other infectious diseases. A new study published in the journal Nature Nanotechnology shows that this inexpensive tool, called Subak, is effective in determining when nucleic acid cleavage occurs, which happens when an enzyme called nuclease breaks down nucleic acids, such as DNA or RNA, into smaller fragments. The traditional method for identifying nuclease activity, the Fluorescence Resonance Energy Transfer (FRET) probe, is 62 times more expensive to produce than the Subak reporter.

Researchers are developing lipid nanoparticles that may target the lungs. The particles are made of molecules that contain two parts: a positively charged head group and a long lipid tail. The positive charge of the head group helps the particles interact with negatively charged mRNA, and also helps the mRNA escape from cellular structures that engulf the particles once they enter the cells. In tests on mice, the researchers showed that they could use the particles to deliver mRNA encoding CRISPR/Cas9 components designed to turn off a genetically encoded stop signal in the animals' lung cells. When this stop signal is removed, a gene for a fluorescent protein lights up. Measuring this fluorescent signal allows researchers to determine what percentage of cells have successfully expressed the mRNA. After one dose of mRNA, about 40% of lung epithelial cells were transfected, the researchers found. Two doses brought the level to more than 50% and three doses to 60%. The most important targets for treating lung disease are two types of epithelial cells called club cells and hair cells, and each was transfected at about 15%. These particles could offer an inhalable treatment for cystic fibrosis and other lung diseases. 

A new approach to CRISPR-Cas9 technology allows scientists to introduce particularly long DNA sequences at precise locations in cell genomes at remarkably high efficiencies without the viral delivery systems traditionally used to transport DNA into cells. The researchers found that high levels of double-stranded DNA template can be toxic to cells, resulting in low efficiency. The team knew that single-stranded DNA was less toxic to cells, even at relatively high concentrations. The researchers therefore describe in the paper published in the journal Nature Biotechnology, a method for attaching the modified Cas9 enzyme to a single-stranded template DNA, by adding just a small overhang of double-stranded DNA at the ends. The single-stranded template DNA could more than double the efficiency of gene editing compared to the old double-stranded approach. In addition, the double-stranded ends of the molecules allow the researchers to use Cas9 to enhance the delivery of non-viral vectors into cells. In the study, the researchers used the new DNA template to generate more than one billion CAR-T cells targeting multiple myeloma and showed that their approach could, for the first time, replace two genes associated with rare genetic immune diseases, IL2RA and CTLA4, in their entirety

Discovering the function of a gene requires cloning a DNA sequence and expressing it. Until now, this was performed on a one-gene-at-a-time basis, causing a bottleneck. Scientists at Rutgers University-New Brunswick in collaboration with Johns Hopkins University and Harvard Medical School have invented a technology to clone thousands of genes simultaneously and create massive libraries of proteins from DNA samples, potentially ushering in a new era of functional genomics.

In this work, using human cultured cells, the authors demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ~40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

In this report, the scientists present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. These findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.

CRISPR/Cas9 method can lead to unintended DNA mutations that can have negative effects. Recently, Japanese researchers have developed a new gene-editing technique that is as effective as CRISPR/Cas9, yet significantly reduces these unintended mutations. In a new study published in Nature Communications , researchers led by Osaka University have introduced a new technique called NICER, based on the creation of several small cuts in single DNA strands by an enzyme  Cas 9 nickase. For their first experiments, the research team used human lymphoblastic cells with a known heterozygous mutation in a gene called TK1. When these cells were treated with nickase to induce a single cut in the TK1 region, TK1 activity was recovered at a low rate. However, when nickase induced multiple cuts in this region on both homologous chromosomes, the efficiency of gene correction was increased approximately seventeen-fold via activation of a cellular repair mechanism. Because the NICER method does not involve DNA double-strand breaks or the use of exogenous DNA, this technique appears to be a safe alternative to conventional CRISPR/Cas9 methods. 

Despite its phylogenetic placement with DNA-targeting nucleases, Cas12a2 targets and cleaves RNA. When researchers put Cas12a2 in test tubes with pure DNA and a guide, nothing happened, because its target was not present and it remained inactive. However, sometimes a small amount of activating RNA was present, contaminating the sample. When the RNA was present and Cas12a2 recognized its target, it destroyed all nucleic acids in the tube. Unlike the CRISPR-Cas9 system, when Cas12a2 finds its target, the infected cell(s) die. This mechanism is known as abortive infection. One of the obvious applications of Cas12a2 is in CRISPR diagnostics. It can easily be reprogrammed to detect certain targets, such as RNA viruses, with high specificity. The researchers have already demonstrated the feasibility of this approach in their recent work. Cas12a2 could also be programmed to kill specific cell types, such as tumor cells, for therapeutic purposes. 

The human genome is made up of 40% of regulatory elements that control gene expression. Understanding the function of these regulatory regions is very important for understanding the cause of certain diseases. The study of these regions is complicated because they must be in the context of their genomic and tissue environment and their developmental timeline. To overcome this, researchers at the Max Delbrück Center for Molecular Medicine in Berlin introduced various large-scale mutations using CRISPR-Cas9 in the form of deletions or insertions into the genomes of thousands of C. elegans worms and then monitored the physiological effect of these mutations. In addition, one of the team's bioinformatics researchers developed sequencing software called CRISPR- Downstream Analysis and Reporting Tool (DART), to analyze the generated data. One of their results was the identification of the function of two let-7 microRNA binding sites that work independently in the downstream regulatory region of a gene called lin-41. They were able to show that if one of the two sites were intact, the worms grew normally, otherwise gene expression was incorrect and the worms grew poorly and died.

Here, the scientists describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

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

This article reports the application of RNA nanotechnology for specific and efficient delivery of anti-miRNA seed-targeting sequence to block the growth of prostate cancer in mouse models. Utilizing the thermodynamically ultra-stable three-way junction of the pRNA of phi29 DNA packaging motor, RNA nanoparticles were constructed by bottom-up self-assembly containing the anti-prostate-specific membrane antigen (PSMA) RNA aptamer as a targeting ligand and anti-miR17 or anti-miR21 as therapeutic modules. The 16 nm RNase-resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little or no accumulation in healthy organs 8 hours postinjection, and subsequently repressed tumor growth at low doses with high efficiency.

The past decade has brought rapid and significant innovations in genome-editing techniques. For the first time researchers have the opportunity to manipulate essentially any gene in a plethora of cells and organisms, using targeted nucleases that were designed for sequence-specific binding of the DNA.