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Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4–7 d and obtain prime-edited rice or wheat plants within 3 months.

Precursor-mRNAs (pre-mRNA) can be processed into one or more mature mRNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intron-mediated enhancement (IME), whereas alternative splicing (AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsisgenes from the activated GT form to the inactive AT form. Silencing the AS of HAB1.1(encoding a type 2C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31Arevealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly, altering the constitutive splicing of Act2via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We developed two variants, MAD7-RR and MAD7-RVR, that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5′-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.

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 ZmALS1and 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.

The pulse is a key biomedical signal containing various human physiological and pathological information highly related to cardiovascular diseases. Pulse signals are often collected from the radial artery based on Traditional Chinese Medicine, or by using flexible pressure sensors. However, the wrist wrapped with a flexible pressure sensor exhibits unstable signals under hand motion because of the concave surface of the wrist. By contrast, fingertips have a convex surface and therefore show great promises in stable and long‐term pulse monitoring. Despite the promising potential, the fingertip pulse signal is weak, calling for highly sensitive detecting devices. Here, a highly sensitive and flexible iontronic pressure sensor with a linear sensitivity of 13.5 kPa−1, a swift response, and remarkable stability over 5000 loading/unloading cycles is developed. This sensor enables stable and high‐resolution detection of pulse waveform under both static condition and finger motion. Fingertip pulse waveforms from subjects of different genders, age, and health conditions are collected and analyzed, suggesting that fingertip pulse information is highly similar to that of the radial artery. This work justifies that fingertip is an ideal platform for pulse signals monitoring, which would be a competitive alternative to existing complex health monitoring systems. A highly sensitive and flexible iontronic pressure sensor is developed for monitoring fingertip pulse which is otherwise too weak to be detected by most existing devices. The sensor presents a linear and high sensivitivity of 13.5 kPa−1. This work shows that the fingertip is a more ideal platform than the radial artery for reliable pulse monitoring.