Prime editors, which are CRISPR-Cas9 nickase (H840A)reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%. Introduction of genome modifications such as substitutions, insertions, and deletions that improve agronomic traits can accelerate crop improvement and breeding 1,2. In plants, nuclease-initiated homology-directed repair (HDR) is limited by low efficiency and the difficulty of DNA template delivery 3-6. Cytosine and adenine base editors (CBEs and ABEs) install C•G-to-T•A and A•T-to-G•C transitions 7-9 , and have been successfully used in plants 4. However, base editors are unable to install transversions, insertions, or deletions 7-10. Prime editing uses engineered Cas9 nickase-reverse transcriptase (RT) fusion proteins paired with a pegRNA that encodes the desired edit 11. The RT domain uses a nicked genomic DNA strand as a primer for the synthesis of an edited DNA flap templated by an extension on the pegRNA. Subsequent DNA repair incorporates the edited flap, permanently installing the programmed edit 11. To optimize prime editing for plants, we first compared three plant prime editor systems (PPEs): PPE2, PPE3, and PPE3b 11 (Fig. 1a). PPE2 consists of a nCas9(H840A) fused to an engineered M-MLV RT, and a pegRNA composed of a primer binding site (PBS) and an RT template 11. PPE3 adds an additional nicking single guide RNA (sgRNA) to cleave the non-edited strand, which facilitates favorable DNA repair. In PPE3b, this nicking sgRNA targets the edited sequence, thereby preventing nicking of the non-edited strand until after editing occurs, resulting in fewer indels in mammalian cells 11. We codon-optimized PPE genes for cereal plants and expressed them using the maize Ubiquitin-1 (Ubi-1) promoter (Fig. 1b). We used the OsU3 (or TaU6) and TaU3 promoters to drive pegRNA and nicking sgRNA transcription, respectively. To test whether other RTs support prime editing, we replaced the engineered M-MLV RT with either the CaMV RT (RT-CaMV) from cauliflower mosaic virus 12 or a retron-derived RT (RT-retron) from E. coli BL21 (ref. 13) (Fig. 1b). We first used our previously described 14 rice protoplast reporter system to test the PPE system for blue fluorescent protein (BFP) to green fluorescent protein (GFP) conversion, which requires changing codon 66 from CAC (histidine) to TAC (tyrosine)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems have been successfully used as efficient tools for genome editing in a variety of species. We used the CRISPR/Cas9 system to mutate the Gn1a (Os01g0197700), DEP1 (Os09g0441900), GS3 (Os03g0407400), and IPA1 (Os08g0509600) genes of rice cultivar Zhonghua 11, genes which have been reported to function as regulators of grain number, panicle architecture, grain size and plant architecture, respectively. Analysis of the phenotypes and frequencies of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in inducing targeted gene editing, with the desired genes being edited in 42.5% (Gn1a), 67.5% (DEP1), 57.5% (GS3), and 27.5% (IPA1) of the transformed plants. The T2 generation of the gn1a, dep1, and gs3 mutants featured enhanced grain number, dense erect panicles, and larger grain size, respectively. Furthermore, semi-dwarf, and grain with long awn, phenotypes were observed in dep1 and gs3 mutants, respectively. The ipa1 mutants showed two contrasting phenotypes, having either fewer tillers or more tillers, depending on the changes induced in the OsmiR156 target region. In addition, we found that mutants with deletions occurred more frequently than previous reports had indicated and that off-targeting had taken place in highly similar target sequences. These results proved that multiple regulators of important traits can be modified in a single cultivar by CRISPR/Cas9, and thus facilitate the dissection of complex gene regulatory networks in the same genomic background and the stacking of important traits in cultivated varieties.

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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.

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.

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Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.