CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting cleaving single-stranded RNA (ssRNA) molecules the phage genome. Here, we employ to engineer interference with an virus, Turnip Mosaic Virus (TuMV), plants. produces green fluorescent protein (GFP)-expressing TuMV transient assays stable overexpression lines Nicotiana benthamiana. CRISPR (crRNAs)...

Although CRISPR-Cas9 has revolutionized our ability to generate site-specific double-strand breaks, precise editing of the genome remains challenging in most eukaryotes, including plants (Shan et al., 2013). In plants, homology-directed repair is inefficient, limiting make edits DNA sequence (Ali 2020; Butt 2017). Moreover, cytosine and adenine base editors have serious drawbacks lower efficiency, unclean edited possibility off-target mutations at other loci (Rees Liu, 2018). Chimeric...

Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant architecture, known their role in inhibiting shoot branching (tillering).We used CRISPR/Cas9 rice (Oryza sativa) disruption CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls step SL biosynthesis. The ccd7 mutants exhibited striking increase tillering, combined with reduced height,...

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based platform in plants evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance splicing inhibitors. mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of Studies structural basis inhibitor binding SGRs corroborate phenotype. This can be used interrogate molecular functions key biomolecules engineer...

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, gene editing is inefficient and requires the simultaneous delivery of a DNA template homology-directed repair (HDR). Here, we used to generate double-strand breaks deliver an RNA HDR rice (Oryza sativa). We chimeric single-guide (cgRNA) molecules carrying both sequences target site specificity (to breaks) direct HDR), flanked by regions homology target. Gene was more efficient...

Abstract Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric combines functions of Cas9, which produces targeted specific DNA double-strand...

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA DNA viruses. CRISPR/Cas9 has broad applications in basic research biotechnology been widely used across eukaryotic for genome engineering functional analysis of genes. The recently developed CRISPR/Cas13 target rather than thus offer new potential transcriptome combatting Here, we CRISPR/LshCas13a to stably engineer...

Abstract Genome-editing technologies have revolutionized research in plant biology, with major implications for agriculture and worldwide food security, particularly the face of challenges such as climate change increasing human populations. Among these technologies, clustered regularly interspaced short palindromic repeats [CRISPR]–CRISPR-associated protein [Cas] systems are now widely used editing crop genomes. In this review, we provide an overview CRISPR–Cas technology its most...

Plant development is highly adaptable and controlled by a combination of various regulatory circuits that integrate internal environmental cues. The phytohormone auxin mediates such growth responses, acting as dynamic signal in the control morphogenesis via coordinating interplay between cell cycle progression differentiation. Mutants chromatin-remodeling component PROPORZ1 (PRZ1; also known AtADA2b) are impaired effects on morphogenesis, suggestive an involvement PRZ1-dependent chromatin...

Summary Alternative splicing ( AS ) of precursor RNA s enhances transcriptome plasticity and proteome diversity in response to diverse growth stress cues. Recent work has shown that is pervasive across plant species, with more than 60% intron‐containing genes producing different isoforms. Mammalian cell‐based assays have discovered various inhibitors . Here, we show the macrolide pladienolide B PB inhibits constitutive plants. Also, our sequencing ‐seq) data revealed mimics abiotic signals...

Abstract Pigmented rice ( Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies 5 varieties evaluated the genetic variation among 51 by resequencing an additional 46 varieties. Phylogenetic analyses divided into four varietal groups: Geng-japonica , Xian-indica circum- Aus Basmati. Metabolomics ionomics profiling revealed that black are in aromatic secondary metabolites. We established...

Abiotic stresses profoundly affect plant growth and development limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich factors play key role in pre-mRNA to regulate different biological processes under stress conditions. Alternative (AS) SR transcripts other stress-responsive genes generates multiple splice isoforms contribute protein diversity, modulate expression, tolerance. Here, we investigated...

Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity proteome; these precisely regulated processes also fine-tune responses to cues related growth, development, stresses. Small-molecule inhibitors that perturb provide invaluable tools for use as chemical probes uncover molecular underpinnings regulation potential anticancer compounds. Here, we show herboxidiene (GEX1A) inhibits both constitutive splicing. Moreover, GEX1A activates genome-wide...

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits interest in diverse species; however, this technique has rarely been applied plants. Here, we designed, built, tested chimeric fusion T7 RNA Polymerase (RNAP) deaminase to enable target interest. We our RNAP-DNA base editor <i>Nicotiana benthamiana</i> transient assays transgene...

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription pre-mRNA splicing. Alternative splicing increases coding potential genome from multiexon genes regulates through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family factors, are key players alternative regulate under stress conditions. The rice (Oryza sativa) encodes 22 SR proteins categorized into six subfamilies....

Recent years have provided us with spectacular insights into the biology of plant hormone auxin, leaving impression a highly versatile molecule involved in virtually every aspect development. A combination genetics, biochemistry, and cell has established auxin signaling pathways, leading to identification two distinct modes perception downstream regulatory cascades. Major targets these modules are components polar transport machinery, mediating directional distribution phytohormone...

Abstract The SF3B complex, a multiprotein component of the U2 snRNP spliceosome, plays crucial role in recognizing branch point sequence and facilitates spliceosome assembly activation. Several chemicals that bind SF3B1 PHF5A subunits complex inhibit splicing. We recently generated splicing inhibitor-resistant mutant named S F3B1 G EX1A R ESISTANT 4 ( SGR4 ) using CRISPR-mediated directed evolution, whereas O verexpression-PHF5A esistance , OGR was by expressing an engineered version...

Plants employ sophisticated molecular machinery to fine-tune their responses growth, developmental, and stress cues. Gene expression influences plant cellular through regulatory processes such as transcription splicing. Pre-mRNA is alternatively spliced increase the genome coding potential further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize cis-elements both constitutive alternative Several studies have reported SR protein genes...

Abstract Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses bacteria. have been harnessed for the overproduction ssDNA, genome engineering directed evolution bacteria, yeast mammalian cells. Retron-mediated ssDNA production plants could unlock their potential applications plant biotechnology. For example, can be used as template homology-directed repair (HDR) several organisms. However, current gene editing...

RNA-directed DNA methylation (RdDM) is essential for de novo in higher plants, and recent reports established novel elements of this silencing pathway the model organism Arabidopsis thaliana. INVOLVED IN DE NOVO METHYLATION 2 (IDN2) closely related FACTOR OF (FDM) are members a plant-specific family dsRNA-binding proteins characterized by conserved XH/XS domains implicated regulation RdDM at chromatin targets. Genetic analyses have suggested redundant as well non-overlapping activities...

Abstract CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting cleaving single stranded RNA (ssRNA) molecules the phage genome. Here, we employ to engineer interference with an virus, Turnip Mosaic Virus (TuMV), plants. produced green fluorescent protein (GFP) expressing TuMV transient assays stable overexpression lines Nicotiana benthamiana . crRNAs...

Directional transport of auxin is essential for plant development, with PIN proteins representing an integral part the machinery that controls hormone distribution. However, unlike rapidly emerging framework molecular determinants regulating protein abundance and subcellular localization, insights into mechanisms controlling transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), evolutionary conserved plant-specific DNA binding acts on genes. Consistent...