RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

RNA targeting with CRISPR-Cas13.

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1111&context=biochemfacpub

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

/pdf/a-crispr-cas9-toolkit-for-multiplex-genome-editing-in-plants-1oc3gjq4qb.pdf

A CRISPR/Cas9 toolkit for multiplex genome editing in plants

Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Plants depend on cell-autonomous innate immune mechanisms for protection against infection and these pathways are activated in response to pattern recognition receptor (PRR) engagement. However, as is the case in mammals, PRR signalling in plants must be tightly controlled to avoid pathological outcomes; this Review focuses on the mechanisms that regulate plant PRR signalling.

Regulation of pattern recognition receptor signalling in plants.

Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue. Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts. We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

/pdf/a-highly-efficient-rice-green-tissue-protoplast-system-for-3fbvvg981j.pdf

A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif–containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

Microbe-associated molecular pattern (MAMP)-triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif-containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor-like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN-triggered immunity in rice. Silencing of OsCERK1 suppressed PGN-induced (and chitin-induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor-binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand-induced heterooligomeric receptor complexes. Furthermore, the receptor-like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.12710

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity.

The receptor-like cytoplasmic kinase, OsRLCK176 has been reported to participate in both chitin- and PGN-induced immunity in rice. Here, we further researched the function of the homologous proteins, OsRLCK57, OsRLCK107 and OsRLCK118, in chitin- and PGN immunity in rice. Silencing of OsRLCK57,OsRLCK107 and OsRLCK118 suppressed chitin- and PGN-induced immunity responses, including reactive oxygen species generation, defense gene expression. Furthermore, OsRLCK107 could interact with OsCERK1 in a MAMP induced way, which suggested a possible physiological relevance of OsRLCKs107 to OsCERK1 pathway. OsRLCK57, OsRLCK107 and OsRLCK118, positively regulate chitin- and PGN- induced responses in rice, similar to that observed in OsRLCK176.

/pdf/osrlck-57-osrlck107-and-osrlck118-positively-regulate-chitin-iiyazgp7xs.pdf

OsRLCK 57, OsRLCK107 and OsRLCK118 Positively Regulate Chitin- and PGN-Induced Immunity in Rice

Plant innate immunity relies on successful detection of trespassing pathogens through recognizing their microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) at the cell surface. We recently reported two rice lysin motif (LysM)-containing proteins, OsLYP4 and OsLYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Here we further demonstrated the important roles of OsLYP4 and OsLYP6 in rice defense signaling, as silencing of either LYP impaired the defense marker gene activation induced by either bacterial pathogen Xanthomonas oryzaecola or fungal pathogen Magnaporthe oryzae. Moreover, we found that OsLYP4 and OsLYP6 could form homo- and hetero-dimers, and could interact with CEBiP, suggesting an unexpected complexity of chitin perception in rice.

Ying Ao

Papers

A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity.

OsRLCK 57, OsRLCK107 and OsRLCK118 Positively Regulate Chitin- and PGN-Induced Immunity in Rice

OsLYP4 and OsLYP6 play critical roles in rice defense signal transduction