Simultaneous and multiplexed phenotyping of circulating exosomes with the orthogonal CRISPR-Cas platform.

Simultaneous and multiplexed exosome protein profiling via an orthogonal CRISPR-Cas platform was achieved in this work. Aptamers were recruited to translate exosome surface protein information into Cas12a/Cas13a cleavage activity. The established multiplexed platform performed robustly with biological matrixes and could profile exosome proteins in clinical serum samples.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

Split G-quadruplex based PfAgo sensing platform for nucleotide mutation discrimination and human genotyping.

A PfAgo-G4 sensing platform exploiting G4 as a signal reporter was proposed, validated, and optimized. By introducing two mismatches at the Link strand, a universal nucleotide design rule was established for accurate single nucleotide polymorphism discrimination with PfAgo-G4. The FUT2 gene was then successfully and accurately genotyped using human buccal swab samples.

Split G-quadruplex-programmed label-free CRISPR-Cas12a sensing system.

A split G-quadruplex (G4)-programmed Cas12a platform was established, validated, and optimized. The split G4 motif was recruited as substrate for Cas12a, and the label-free sensing platform provided a concentration-dependent response towards the input target. Furthermore, exosomal surface proteins from cultured cancer cells and clinical samples were detected and profiled.

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection.

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 µL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

A crosslinked colloidal network of peptide/nucleic base amphiphiles for targeted cancer cell encapsulation.

The use of peptide amphiphiles (PAs) is becoming increasingly popular, not only because of their unique self-assembly properties but also due to the versatility of designs, allowing biological responsiveness, biocompatibility, and easy synthesis, which could potentially contribute to new drug design and disease treatment concepts. Oligonucleotides, another major functional bio-macromolecule class, have been introduced recently as new functional building blocks into PAs, further enriching the tools available for the fabrication of bio-functional PAs. Taking advantage of this, in the present work, two nucleic base-linked (adenine, A and thymine, T) RGD-rich peptide amphiphiles (NPAs) containing the fluorophores naphthalimide and rhodamine (Nph-A and Rh-T) were designed and synthesized. The two NPAs exhibit distinctive assembly behaviours with spherical (Rh-T) and fibrous (Nph-A) morphologies, and mixing Nph-A with Rh-T leads to a densely crosslinked colloidal network (Nph-A/Rh-T) via mutually promoted supramolecular polymerization via nucleation-growth assembly. Because of the RGD-rich sequences in the crosslinked network, further research on in situ targeted cancer cell (MDA-MB-231) encapsulation via RGD-integrin recognition was performed, and the modulation of cell behaviours (e.g., cell viability and migration) was demonstrated using both confocal laser scanning microscopy (CLSM) imaging and a scratch wound healing assay.

Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig.

Domestic pig (Sus scrofa domestica) is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags (ESTs) have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different non-normalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account.

EST pipeline system: detailed and automated EST data processing and mining.

Expressed sequence tags (ESTs) are widely used in gene survey research these years. The EST Pipeline System, software developed by Hangzhou Genomics Institute (HGI), can automatically analyze different scalar EST sequences by suitable methods. All the analysis reports, including those of vector masking, sequence assembly, gene annotation, Gene Ontology classification, and some other analyses, can be browsed and searched as well as downloaded in the Excel format from the web interface, saving research efforts from routine data processing for biological rules embedded in the data.