Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.

Identification and Analysis of Genes Involved in Double Fertilization in Rice.

Double fertilization is a key determinant of grain yield, and the failure of fertilization during hybridization is one important reason for reproductive isolation. Therefore, fertilization has a very important role in the production of high-yield and well-quality hybrid of rice. Here, we used RNA sequencing technology to study the change of the transcriptome during double fertilization with the help of the mutant fertilization barrier (feb) that failed to finish fertilization process and led to seed abortion. The results showed that 1669 genes were related to the guided growth of pollen tubes, 332 genes were involved in the recognition and fusion of the male-female gametes, and 430 genes were associated with zygote formation and early free endosperm nuclear division. Among them, the genes related to carbohydrate metabolism; signal transduction pathways were enriched in the guided growth of pollen tubes, the genes involved in the photosynthesis; fatty acid synthesis pathways were activated by the recognition and fusion of the male-female gametes; and the cell cycle-related genes might play an essential role in zygote formation and early endosperm nuclear division. Furthermore, among the 1669 pollen tube-related genes, it was found that 7 arabinogalactan proteins (AGPs), 1 cysteine-rich peptide (CRP), and 15 receptor-like kinases (RLKs) were specifically expressed in anther, while 2 AGPs, 7 CRPs, and 5 RLKs in pistil, showing obvious unequal distribution which implied they might play different roles in anther and pistil during fertilization. These studies laid a solid foundation for revealing double fertilization mechanism of rice and for the follow-up investigation.

Identification of Hydroxyproline-Containing Proteins and Hydroxylation of Proline Residues in Rice.

The hydroxyproline-containing proteins (HCPs) among secretory and vacuolar proteins play important roles in growth and development of higher plants. Many hydroxyproline-rich glycoproteins (HRGPs), including Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs), are identified as HCPs by bioinformatics approaches. The experimental evidence for validation of novel proline hydroxylation sites is vital for understanding their functional roles. In this study, the 62 HCPs containing 114 hydroxyproline (O, Hyp) residues were identified, and it was found that hydroxylation of proline residues in the HCPs could either constitute attachment sites for glycans or have other biological function in rice. The glycomodules of AO, OA, OG, VO, LO, and OE were abundant in the 62 HCPs. Further analysis showed that the 22 of 62 HCPs contained both signal peptides and transmembrane domains, and the 19 HCPs only contained transmembrane domains, while 21 HCPs contained neither. This study indicated the feasibility of mass spectrometry-based proteomics combined with bioinformatics approaches for the large-scale characterization of Hyp sites from complex protein digest mixtures. Furthermore, the expression of AGPs in rice was detected by using ß-GlcY reagent and JIM13 antibody. The results displayed that the AGPs were widely distributed in different tissues and organs of rice, especially expressed highly in lateral root, pollen and embryo. In conclusion, our study revealed that the HCPs and Hyp residues in rice were ubiquitous and that these Hyps could be candidates for linking to glycans, which laid the foundation for further studying the functions of HCPs and hydroxylation of proline residues in rice.