Yuanyuan Jiang

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

Phosphate-containing metabolites and alkaline phosphatase (ALP) activity are useful biomarkers for many types of diseases. However, there are few straightforward, sensitive, and efficient colorimetric methods for the quantification of them only when resorting to unstable transition metal ions or specially designed organic substrates. Herein, we have demonstrated that histidine-protected gold nanoclusters (His–AuNCs) possess intrinsic peroxidase-like activity with Au atom facilitated formation of superoxide anions (O2•–) and their electron transfer ability. More interestingly, phosphate-containing metabolites can severely inhibit the peroxidase-like activity of His–AuNCs by blocking the generation of O2•– and electron transfer, and then ALP is able to restore the inhibition process through hydrolyzing the phosphate-containing metabolites. Therefore, using peroxidase-triggered chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) as an amplifier, a colorimetric on–off–on switch has been developed for sensing phosphate-containing metabolites and ALP based on the logical regulation of such deactivation and reactivation processes for the first time. According to the intrinsic mimic enzyme-catalyzed amplification and clear response mechanism, our colorimetric assay exhibits excellent sensitivity, selectivity, and sensing performance. Furthermore, on the basis of the proposed colorimetric sensors, a combinatorial “NOR+IMPLICATION” logic gate is further rationally constructed.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens. The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.