Impact of Chromatin Organization and Epigenetics on CRISPR-Cas and TALEN Genome Editing.

DNA lies at the heart of the central dogma of life. Altering DNA can modify the flow of information in fundamental cellular processes such as transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research.

Harnessing noncanonical crRNA for highly efficient genome editing.

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target.

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression.

Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater.

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.

Automated, self-resistance gene-guided, and high-throughput genome mining of bioactive natural products from .

Natural products (NPs) produced by bacteria, fungi and plants are a major source of drug leads. species are particularly important in this regard as they produce numerous natural products with prominent bioactivities. Here we report a fully a utomated, s calable and high-throughput platform for discovery of bioactive n atural p roducts in (FAST-NPS).

To Cut or not to Cut: Next-generation Genome Editors for Precision Genome Engineering.

Since the original report of repurposing the CRISPR/Cas9 system for genome engineering, the past decade has witnessed profound improvement in our ability to efficiently manipulate the mammalian genome. However, significant challenges lie ahead that hinder the translation of CRISPR-based gene editing technologies into safe and effective therapeutics. The CRISPR systems often have a limited target scope due to PAM restrictions, and the off-target activity also poses serious risks for therapeutic applications.

Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection.

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs.

A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis.

The need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection.

Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers.

CRISPR/Cas9 is a powerful genome editing tool, but its off-target cleavage activity can result in unintended adverse outcomes for therapeutic applications. Here we report the design of a simple tunable CRISPR controller in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Dose-dependent control over Cas9 editing and dCas9 induction was achieved, which drastically improved the specificity and biosafety of the CRISPR/Cas9 system.

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

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 state-of-the-art strategies of protein engineering for enzyme stabilization.

Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently.