Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot.

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00139-7.

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits.

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3-, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16-22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23-28.9%) and biomass by (24.1-29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

A detailed landscape of CRISPR-Cas-mediated plant disease and pest management.

Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.

Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants.

Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200-250 nucleotides long sequence to either 5' or 3' ends of guide RNA did not significantly affect Cas9 cleavage activity.

Comparative transcriptome profiling reveals the basis of differential sheath blight disease response in tolerant and susceptible rice genotypes.

Rice sheath blight (ShB) disease, caused by the fungal pathogen Rhizoctonia solani AG1-IA, is one of the devastating diseases and causes severe yield losses all over the world. No completely resistant germplasm is known till now, and as a result, the progress in resistance breeding is unsatisfactory. Basic studies to identify candidate genes, QTLs, and to better understand the host-pathogen interaction are also scanty. In this study, we report the identification of a new ShB-tolerant rice germplasm, CR 1014. Further, we investigated the basis of tolerance by exploring the disease responsive differentially expressed transcriptome and comparing them with that of a susceptible variety, Swarna-Sub1. A total of 815 and 551 genes were found to be differentially regulated in CR 1014 and Swarna-Sub1, respectively, at two different time points. The result shows that the ability to upregulate genes for glycosyl hydrolase, secondary metabolite biosynthesis, cytoskeleton and membrane integrity, the glycolytic pathway, and maintaining photosynthesis make CR 1014 a superior performer in resisting the ShB pathogen. We discuss several putative candidate genes for ShB resistance. The present study, for the first time, revealed the basis of ShB tolerance in the germplasm CR1014 and should prove to be particularly valuable in understanding molecular response to ShB infection. The knowledge could be utilized to devise strategies to manage the disease better.

Precise plant genome editing using base editors and prime editors.

The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.

Preformed aerenchyma determines the differential tolerance response under partial submergence imposed by fresh and saline water flooding in rice.

Plant's response to fresh- and saline-water flooding and the resulting partial submergence, seems different due to the added complexities of element toxicity of salinity. We identified a few rice genotypes which can tolerate combined stresses of partial submergence and salinity during saline water flooding. To gain mechanistic insights, we compared two rice genotypes: Varshadhan (freshwater-flooding tolerant) and Rashpanjor (both fresh- and saline-water flooding tolerant). We found greater ethylene production and increased "respiratory burst oxidase homolog" (RBOH)-mediated reactive oxygen species (ROS) production led to well-developed constitutive aerenchyma formation in Rashpanjor, which makes it preadapted to withstand fresh- and saline-water flooding. On the contrary, an induced aerenchyma formation-dependent tolerance mechanism of Varshadhan worked well for freshwater flooding but failed to provide tolerance to saline-water flooding. Additional salt stress was found to significantly inhibit the induced aerenchyma formation process due to the dampening of ROS signaling by the action of metallothionein in Varshadhan. Besides, inconspicuous changes in ionic regulation processes in these two genotypes under saline-water flooding suggest preadapted constitutive aerenchyma formation plays a more significant role than elemental toxicity per se in tolerating combined stresses encountered during saline water flooding in rice. Overall, our study indicated that well-developed constitutive aerenchyma provide an adaptive advantage during partial submergence due to saline water flooding in rice as the key process of induced aerenchyma formation is hampered in the presence of salinity stress coupled with partial submergence.

Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica.

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.

Base Editing Landscape Extends to Perform Transversion Mutation.

Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.

Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors.

The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break (DSB) or supplying a DNA donor template for homology-directed repair (HDR). In this study, adenine base editors (ABEs) were used for rapid generation of precise point mutations in two distinct genes, OsWSL5, and OsZEBRA3 (Z3), in both rice protoplasts and regenerated plants. The precisely engineered point mutations were stably inherited to subsequent generations. These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern, respectively. Through selfing and genetic segregation, transgene-free, base edited wsl5 and z3 mutants were obtained in a short period of time. We noticed a novel mutation (V540A) in Z3 locus could also mimic the phenotype of Z3 mutation (S542P). Furthermore, we observed unexpected non- A/G or T/C mutations in the ABE editing window in a few of the edited plants. The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.