Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus (PfAgo) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow PfAgo selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process. The integrated single-tube system achieved great efficiency for enriching rare mutations compared with a divided system separating the cleavage and amplification. Thus, A-Star enables easy detection and quantification of 0.01% rare mutations with ≥5500-fold increase in efficiency. The feasibility of A-Star was also demonstrated for detecting oncogenic mutations in solid tumor tissues and blood samples. Remarkably, A-Star achieved simultaneous detection of multiple oncogenes through a simple single-tube reaction by orthogonal guide-directed specific cleavage. This study demonstrates a supersensitive and rapid nucleic acid detection system with promising potential for both research and therapeutic applications.

/pdf/argonaute-integrated-single-tube-pcr-system-enables-e0xjh2mbat.pdf

Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations

Nucleic acid therapeutics have emerged as a powerful method for treatment of many diseases. However, the challenge lies in safe and efficient delivery of nucleic acids to their target site, as they need to cross various extracellular and intracellular barriers. Mammalian viruses have initially been favored for delivery of nucleic acid therapeutics, but safety concerns regarding their immunogenicity and potential of integration have fueled the search for alternative delivery strategies. For example, chemistry and bioengineering have led to advances in the use of nonviral vectors composed of lipids and other polymers; nevertheless, the synthetic systems often do not match the efficiency achieved using the biological systems. More recently, researchers have turned toward the development of plant viruses and bacteriophages and virus-like particles as an alternative or complementary approach. These systems unite the properties of both the viral and nonviral systems and as such are a new exciting avenue toward nucleic acid delivery. This review highlights the benefits of plant viral and bacteriophage delivery of nucleic acids and provides a summary of the current progress in research in this field. WIREs Nanomed Nanobiotechnol 2018, 10:e1487. doi: 10.1002/wnan.1487

This article is categorized under: 

Biology-Inspired Nanomaterials > Protein and Virus-Based Structures

Plant viral and bacteriophage delivery of nucleic acid therapeutics

In this review, we focus on the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome editing system, in the identification of resistance mechanisms and in overcoming drug resistance in the most frequent solid tumors. Data were collected by conducting systematic searching of scientific English literature using specific keywords such as “cancer”, “CRISPR” and related combinations. The review findings revealed the importance of CRISPR/Cas9 system in understanding drug resistance mechanisms and identification of resistance-related genes such as PBRM1, SLFN11 and ATPE1 in different cancers. We also provided an overview of genes, including RSF1, CDK5, and SGOL1, whose disruption can synergize with the currently available drugs such as paclitaxel and sorafenib. The data suggest CRISPR/Cas9 system as a useful tool in elucidating the molecular basis of drug resistance and improving clinical outcomes.

/pdf/crispr-cas9-for-overcoming-drug-resistance-in-solid-tumors-4jkpc1d3fu.pdf

CRISPR/Cas9 for overcoming drug resistance in solid tumors

The application and progression of CRISPR/Cas9 technology in ophthalmological diseases

Widely used gene editing strategies in cancer treatment a systematic review

Gene editing technology has been at its mature stage with the successful development of TALENs and CRISPR/Cas enzymes. The genetically modified endonucleases of ZFNs, TALENs, and CRISPR/Cas are widely used in the development of genetically modified cells or organisms. Among the enzymes that possess gene editing ability, CRISPR/Cas is the latest member with high efficiency in gene editing and simplicity in cloning. This review discusses the discovery of CRISPR, the development of the CRISPR/Cas system, and its applications as a new gene editing system.

CRISPR/Cas: A Faster and More Efficient Gene Editing System.

A primerless amplification suitable for enrichment of particular genotype cfDNA which is a one-dimensional material has been developed. This primerless amplification coordinated by two thermostable enzymes of endonuclease and proofreading polymerase, functions as a genotype switch in analyzing cfDNA. The endonuclease digests the wild-typed fragments into mega-primer and discriminately destroys the wild-type DNA alleles. The DNA polymerase proofreads the megaprimer and then extends the mega-primer using the mutant DNA as the template. The prototypes of this technology were applied to two hotspot mutations of APC and EGFR with confirmed by DNA sequencing analysis. Genotype switch was then employed to clinical cfDNA assay targeting PIK3CA. Data from the clinical application suggest its potential in early cancer diagnosis.

Primerless Amplification for Circulating Tumor DNA Assays.

The invention discloses a method for detecting tolerance capability of gene mutation by inserting partial wobble oligonucleotide. The method comprises the following steps: adding restriction endonuclease sites on the terminals of synthesized NNT and/or NNC strand nucleic fragments, directly connecting vector fragments subjected to restriction enzyme digestion, introducing vectors into host cells, expressing the host cells to generate a series of mutant nucleic acids with unequal lengths and different sequences, and testing the expression activity of the mutant nucleic acids so as to assess the tolerance capability of gene mutation. The method has the advantages including that whether a tested gene has base use bias can be tested; in addition, whether the insertion length of the tested gene is strictly limited can be tested.

Method for detecting tolerance capability of gene mutation by inserting partial wobble oligonucleotide

Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay.

/pdf/a-mutagenesis-assay-for-reporter-gene-screening-using-1es27w4gvd.pdf

A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

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Papers

CRISPR/Cas: A Faster and More Efficient Gene Editing System.

Primerless Amplification for Circulating Tumor DNA Assays.

Method for detecting tolerance capability of gene mutation by inserting partial wobble oligonucleotide

A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC