In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

A simple and efficient method for betalain quantification in RUBY-expressing plant samples.

The RUBY reporter system has demonstrated great potential as a visible marker to monitor gene expression in both transiently and stably transformed plant tissues. Ectopic expression of the RUBY reporter leads to bright red pigmentation in plant tissues that do not naturally accumulate betalain. Unlike traditional visual markers such as ß-glucuronidase (GUS), luciferase (LUC), and various fluorescent proteins, the RUBY reporter system does not require sample sacrifice or special equipment for visualizing the gene expression. However, a robust quantitative analysis method for betalain content has been lacking, limiting accurate comparative analyses. In this work, we present a simple and rapid protocol for quantitative evaluation of RUBY expression in transgenic plant tissues. Using this method, we demonstrate that differential RUBY expression can be quantified in transiently transformed leaf tissues, such as agroinfiltrated Nicotiana benthamiana leaves, and in stable transgenic maize tissues, including seeds, leaves, and roots. We found that grinding fresh tissues with a hand grinder and plastic pestle, without the use of liquid nitrogen, is an effective method for rapid betalain extraction. Betalain contents estimated by spectrophotometric and High-Performance Liquid Chromatography (HPLC) analyses were highly consistent, validating that our rapid betalain extraction and quantification method is suitable for comparative analysis. In addition, betalain content was strongly correlated with RUBY expression level in agroinfiltrated N. benthamiana leaves, suggesting that our method can be useful for monitoring transient transformation efficiency in plants. Using our rapid protocol, we quantified varying levels of betalain pigment in N. benthamiana leaves, ranging from 110 to 1066 mg/kg of tissue, and in maize samples, ranging from 15.3 to 1028.7 mg/kg of tissue. This method is expected to streamline comparative studies in plants, providing valuable insights into the effectiveness of various promoters, enhancers, or other regulatory elements used in transgenic constructs.

CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution.

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements. [BMB Reports 2024; 57(1): 30-39].

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.

Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.

CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.

BACKGROUND: E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products. RESULTS: The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield. CONCLUSIONS: The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives.

Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.

CRISPR-Mediated Engineering across the Central Dogma in Plant Biology for Basic Research and Crop Improvement.

The central dogma (CD) of molecular biology is the transfer of genetic information from DNA to RNA to protein. Major CD processes governing genetic flow include the cell cycle, DNA replication, chromosome packaging, epigenetic changes, transcription, posttranscriptional alterations, translation, and posttranslational modifications. The CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next generation. Engineering of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world population. CRISPR technology enables programmable editing of CD processes to alter DNA, RNA, or protein, which would have been impossible in the past. Here, an overview of recent advancements in CRISPR tool development and CRISPR-based CD modulations that expedite basic and applied plant research is provided. Furthermore, CRISPR applications in major thriving areas of research, such as gene discovery (allele mining and cryptic gene activation), introgression (de novo domestication and haploid induction), and application of desired traits beneficial to farmers or consumers (biotic/abiotic stress-resilient crops, plant cell factories, and delayed senescence), are described. Finally, the global regulatory policies, challenges, and prospects for CRISPR-mediated crop improvement are discussed.

Salinity Stress in Potato: Understanding Physiological, Biochemical and Molecular Responses.

Among abiotic stresses, salinity is a major global threat to agriculture, causing severe damage to crop production and productivity. Potato (Solanum tuberosum) is regarded as a future food crop by FAO to ensure food security, which is severely affected by salinity. The growth of the potato plant is inhibited under salt stress due to osmotic stress-induced ion toxicity. Salinity-mediated osmotic stress leads to physiological changes in the plant, including nutrient imbalance, impairment in detoxifying reactive oxygen species (ROS), membrane damage, and reduced photosynthetic activities. Several physiological and biochemical phenomena, such as the maintenance of plant water status, transpiration, respiration, water use efficiency, hormonal balance, leaf area, germination, and antioxidants production are adversely affected. The ROS under salinity stress leads to the increased plasma membrane permeability and extravasations of substances, which causes water imbalance and plasmolysis. However, potato plants cope with salinity mediated oxidative stress conditions by enhancing both enzymatic and non-enzymatic antioxidant activities. The osmoprotectants, such as proline, polyols (sorbitol, mannitol, xylitol, lactitol, and maltitol), and quaternary ammonium compound (glycine betaine) are synthesized to overcome the adverse effect of salinity. The salinity response and tolerance include complex and multifaceted mechanisms that are controlled by multiple proteins and their interactions. This review aims to redraw the attention of researchers to explore the current physiological, biochemical and molecular responses and subsequently develop potential mitigation strategies against salt stress in potatoes.

Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era.

Plants and microbes are co-evolved and interact with each other in nature. Plant-associated microbes, often referred to as plant microbiota, are an integral part of plant life. Depending on the health effects on hosts, plant-microbe (PM) interactions are either beneficial or harmful. The role of microbiota in plant growth promotion (PGP) and protection against various stresses is well known. Recently, our knowledge of community composition of plant microbiome and significant driving factors have significantly improved. So, the use of plant microbiome is a reliable approach for a next green revolution and to meet the global food demand in sustainable and eco-friendly agriculture. An application of the multifaceted PM interactions needs the use of novel tools to know critical genetic and molecular aspects. Recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) tools are of great interest to explore PM interactions. A systematic understanding of the PM interactions will enable the application of GE tools to enhance the capacity of microbes or plants for agronomic trait improvement. This review focuses on applying GE techniques in plants or associated microbiota for discovering the fundamentals of the PM interactions, disease resistance, PGP activity, and future implications in agriculture.