The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system’s specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings.

Though plenty of viral and non-viral methods have been rapidly developed for the delivery of the CRISPR/Cas9 system, the direct delivery of Cas9 ribonucleoproteins (RNPs) into the nucleus for genome editing via the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, especially the latter one, remains a challenge. Here we report a delivery vehicle achieved by encapsulating red fluorescent protein (RFP) within chitosan (CS), which can simultaneously deliver engineered Cas9 RNPs with a poly-glutamate peptide tag (E-tag) and DNA donors into a range of cell types with high genome editing efficacy and non-cytotoxicity.

Backgrounds: Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused onSaccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L.Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineerPichia pastoriswith minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L.Conclusions: The research promotes the application ofP. pastorisas a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.

The CRISPR-Cas nuclease has emerged as a powerful genome-editing tool in recent years. The CRISPR-Cas system induces double-strand breaks that can be repaired via the non-homologous end joining or homology-directed repair (HDR) pathway. Compared to non-homologous end joining, HDR can be used for the treatment of incurable monogenetic diseases. Therefore, remarkable efforts have been dedicated to enhancing the efficacy of HDR. In this review, we summarize the currently used strategies for enhancing the HDR efficiency of CRISPR-Cas systems based on three factors: (1) regulation of the key factors in the DNA repair pathways, (2) modulation of the components in the CRISPR machinery, and (3) alteration of the intracellular environment around double-strand breaks. Representative cases and potential solutions for further improving HDR efficiency are also discussed, facilitating the development of new CRISPR technologies to achieve highly precise genetic manipulation in the future.