Genetic Engineering Publications - GEG Tech top picks

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. 

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. 

The standard CAR T cell strategy would be problematic when directed against heart failure or other fibrotic diseases in humans.

Fibroblasts have a normal and important function in the body, particularly in wound healing. CAR T cells that are genetically reprogrammed to attack fibroblasts could survive in the body for months or even years, suppressing the fibroblast population and impairing wound healing for all that time. Therefore, in the new study published in Science, Epstein and colleagues designed a technique for a more temporary and controllable, and much more procedurally simple, type of CAR T cell therapy. They designed an mRNA that encodes a T-cell receptor targeting activated fibroblasts and encapsulated the mRNA in tiny bubble-like lipid nanoparticles, which are themselves coated with molecules that lodge in T cells. Injections of this therapy into mice modeling heart failure successfully reprogrammed a large population of mouse T cells, causing a major reduction in cardiac fibrosis in the animals and restoration of a mostly normal heart size and function with no sign of continued anti-fibroblast T cell activity one week after treatment. Researchers continue to test this mRNA-based transient CAR T-cell technology, with the hope of eventually starting clinical trials

Cas9 can be implemented as RNA-targeting system to track mRNA in living cells. Nuclear export is enabled by efficient targeting of GFP-fused Cas9 to an endogenous mRNA. The approach provides a new and versatile platform for RNA-targeting with applications in RNA imaging and beyond.

Here, the scientists develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes.

Immunotherapy is an essential component of cancer treatment. However, despite the success of immunotherapies for various types of cancer, only a few cancer patients have shown responses to current immune therapies and therapies can have adverse effects. Therefore, researchers have created intramuscular messenger ribonucleic acid (mRNA) lipid nanoparticle (LNP) vaccines as likely candidates for therapeutic cancer vaccines. RNA molecules can be encapsulated in LNPs. In addition, single-stranded mRNA can encode tumor vaccine neo-antigens, while small double-stranded interfering RNA can encode knockdown checkpoint inhibitors to adjust immune responses through RNA-induced activation of suppressed immune cells. Circular-type RNA can increase expression time, which benefits the generation of chimeric antigen receptors (CAR) in vivo and vaccine antigens. Targeted delivery of LNP in vivo would be essential for generating CAR-T macrophages and lymphocytes, in vivo. Based on the results of the study, the LNP mRNA vaccine platform could be used to develop next-generation personalized cancer vaccines. However, additional research is needed to further our understanding of cancer biology and improve vaccine design for faster clinical translation. 

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. 

Here scientists report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5′ end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators.

In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. the chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. The approach described here provides a universal means of cell type–specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.

Here, the scientists investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. They find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex.

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.

For first time, scientists use CRISPR-Cas9 to target RNA in live cells. They show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation ofACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. They also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Their results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.

Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)–transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency

Silas et al. discover that certain classes of the Cas1gene are fused to a reverse transcriptase gene (RT-Cas1) (see the Perspective by Sontheimer and Marraffini). These RT-Cas1 proteins are able to capture and directly incorporate both DNA and RNA into CRISPR loci. RT-Cas1 systems could be effective against parasitic RNA species, or even to modulate bacterial gene expression.