Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

MiRiQ Database: A Platform for In Silico Rice Mutant Screening.

Genetic studies using mutant resources have significantly contributed to elucidating plant gene function. Massive mutant libraries sequenced by next-generation sequencing technology facilitate mutant identification and functional analysis of genes of interest. Here, we report the creation and release of an open-access database (https://miriq.agr.kyushu-u.ac.jp/index.php), called Mutation-induced Rice in Kyushu University (MiRiQ), designed for in silico mutant screening based on a whole-genome-sequenced mutant library. This database allows any user to easily find mutants of interest without laborious efforts such as large-scale screening by PCR. The initial version of the MiRiQ database (version 1.0) harbors a total of 1.6 million single-nucleotide variants (SNVs) and InDels of 721 M1 plants that were mutagenized by N-methyl-N-nitrosourea treatment of the rice cultivar Nipponbare (Oryza sativa ssp. japonica). The SNVs were distributed among 87% of all 35,630 annotated protein-coding genes of the Nipponbare genome and were predicted to induce missense and nonsense mutations. The MiRiQ database provides built-in tools, such as a search tool by keywords and JBrowse for mutation searches. Users can request mutant seeds in the M2 or M3 generations from a request form linked to this database. We believe that the availability of a wide range of gene mutations in this database will benefit the plant science community and breeders worldwide by accelerating functional genomic research and crop improvement.

Effectively controlled microfluidic trap for spatiotemporal analysis of the electrotaxis of Caenorhabditis elegans.

C. elegans is a popular model organism with a well-developed neural network. Approximately 60% of the genes in C. elegans have genomic counterparts in humans, including those involved in building neural circuits. Therefore, we can extend the study of human neural network mechanisms to C. elegans which is easy to genetically manipulate. C. elegans shows behavioural responses to various external physical and chemical stimuli. Electrotaxis is one of its distinct behavioural responses, which is defined as movement towards the cathode in an electric field. In this study, we developed an effective microfluidic trap system for analysing electrotaxis in C. elegans. In addition, two mutant strains (unc-54(s74) and unc-6(e78)) from wild-type (N2) worms were screened using the system. Wild-type (N2) worms and the two mutant strains clearly showed different behavioural responses to the applied electric field, thus enabling the effective screening of the mutant worms from the wild type (N2). This microfluidic system can be utilized as a platform for the study of behavioural responses, and for the sorting and mutant screening of C. elegans.

The CRISPR/Cas9 system and its applications in crop genome editing.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.

[Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants].

OBJECTIVE: To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53. METHODS: The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants. RESULTS: The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully. CONCLUSION: pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency.

To cope with manganese (Mn) deficiency, plants have evolved an efficient transport system to uptake and redistribute Mn. However, the underlying molecular mechanisms remain to be demonstrated. We carried out a forward genetic screen in a root high-affinity Mn transporter nramp1 mutant background in Arabidopsis thaliana and identified an uncharacterized Mn transport NRAMP2. We investigated the effect of nramp2 mutation on root growth and reactive oxygen species (ROS) accumulation and we also examined the NRAMP2 expression pattern, and the subcellular localization and transport activity of NRAMP2. Mutation of NRAMP2 impaired plant growth, while overexpression of NRAMP2 improved plant growth under low Mn conditions. In the nramp2-1nramp1 double mutant, Mn deficiency inhibited root cell elongation and root hair development, which was associated with increased hydrogen peroxide (H2 O2 ) accumulation. NRAMP2 is preferentially localized to the trans-Golgi network. NRAMP2 has Mn influx transport activity in yeast, and mutation of NRAMP2 led to greater Mn retention in roots. Our results suggest that under Mn-deficient conditions, increased accumulation of H2 O2 is partially responsible for the root growth inhibition and NRAMP2 is involved in remobilization of Mn in Golgi for root growth.

Genome editing in diatoms: achievements and goals.

Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.

Progress in the microbial production of S-adenosyl-L-methionine.

S-Adenosyl-L-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.

High-throughput transformation method for Yarrowia lipolytica mutant library screening.

As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.

Toxic potentiality of bio-oils, from biomass pyrolysis, in cultured cells and Caenorhabditis elegans.

Bio-oils, which are multicomponent mixtures, were produced from two different biomass (rice straw (rice oil) and sawdust of oak tree (oak oil)) by using the slow pyrolysis process, and chemical compositional screening with GC-MS detected several hazardous compounds in both bio-oil samples. The two bio-oils vary in their chemical compositional nature and concentrations. To know the actual hazard potentialities of these bio-oils, toxicological assessments were carried out in a comparative approach by using in vitro (Jurkat T and HepG2 cell) as well as in vivo (Caenorhabditis elegans) systems. A dose-dependent increase in cytotoxicity, cell death (apoptosis), and genotoxicity were observed in cultured cell systems. Similarly, the in vivo system, C. elegans also displayed a dose-dependent decrease in survival. It was found that in comparison with rice oil, oak oil displayed higher toxicity to all models systems, and the susceptibility order of the model systems were Jurkat T > HepG2 > C. elegans. Pursuing the study further toward the underlying mechanism by exploiting the C. elegans mutants screening assay, the bio-oils seem to mediate toxicity through oxidative stress and impairment of immunity. Taken together, bio-oils compositions mainly depend on the feedstock used and the pyrolysis conditions which in turn modulate their toxic potentiality.

A collection of novel Lotus japonicus LORE1 mutants perturbed in the nodulation program induced by the Agrobacterium pusense strain IRBG74.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5´UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s).

Multi-generation study of heavy ion beam-induced mutations and agronomic trait variations to accelerate rice breeding.

Heavy ion beam (HIB) is an effective physical mutagen that has been widely used in plant mutational breeding. Systemic knowledge of the effects caused by different HIB doses at developmental and genomic levels will facilitate efficient breeding for crops. Here we examined the effects of HIB systematically. Kitaake rice seeds were irradiated by ten doses of carbon ion beams (CIB, 25 - 300 Gy), which is the most widely used HIB. We initially examined the growth, development and photosynthetic parameters of the M1 population and found that doses exceeding 125 Gy caused significant physiological damages to rice. Subsequently, we analyzed the genomic variations in 179 M2 individuals from six treatments (25 - 150 Gy) via whole-genome sequencing (WGS). The mutation rate peaks at 100 Gy (2.66×10-7/bp). Importantly, we found that mutations shared among different panicles of the same M1 individual are at low ratios, validating the hypothesis that different panicles may be derived from different progenitor cells. Furthermore, we isolated 129 mutants with distinct phenotypic variations, including changes in agronomic traits, from 11,720 M2 plants, accounting for a 1.1% mutation rate. Among them, about 50% possess stable inheritance in M3. WGS data of 11 stable M4 mutants, including three lines with higher yields, reveal their genomic mutational profiles and candidate genes. Our results demonstrate that HIB is an effective tool that facilitates breeding, that the optimal dose range for rice is 67 - 90% median lethal dose (LD50), and that the mutants isolated here can be further used for functional genomic research, genetic analysis, and breeding.

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin).

Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (ß-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

Applications of flow cytometry sorting in the pharmaceutical industry: A review.

The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures.

A Method Enabling Comprehensive Isolation of Arabidopsis Mutants Exhibiting Unusual Root Mechanical Behavior.

Root penetration into soils is fundamental for land plants to support their own aboveground parts and forage water and nutrients. To elucidate the molecular mechanisms underlying root mechanical penetration, mutants defective in this behavior need to be comprehensively isolated; however, established methods are currently scarce. We herein report a method to screen for these mutants of Arabidopsis thaliana and present their phenotypes. We isolated five mutants using this method, tentatively named creep1 to creep5, the primary roots of which crept over the surface of horizontal hard medium that hampered penetration by the primary root of the wild type, thereby forcing it to spring up on the surface and die. By examining root skewing, which is induced by a touch stimulation that is generated as the primary roots grow along a vertical impenetrable surface, the five creep mutants were subdivided into three groups, namely mutants with the primary root skewing leftward, those skewing rightward, and that growing dispersedly. While the majority of wild type primary roots skewed slightly leftward, nearly half of the primary roots of creep1 and creep5 skewed rightward as viewed from above. The primary roots of creep4 displayed scattered growth, while those of creep2 and creep3 showed a similar phenotype to the wild type primary roots. These results demonstrate the potential of the method developed herein to isolate various mutants that will be useful for investigating root mechanical behavior regulation not only in Arabidopsis, but also in major crops with economical value.

Molecular fingerprints of conazoles via functional genomic profiling of Saccharomyces cerevisiae.

Conazoles were designed to inhibit ergosterol biosynthesis. Conazoles have been widely used as agricultural fungicides and are frequently detected in the environment. Although conazoles have been reported to have adverse effects, such as potential carcinogenic effects, the underlying molecular mechanisms of toxicity remain unclear. Here, the molecular fingerprints of five conazoles (propiconazole (Pro), penconazole (Pen), tebuconazole (Teb), flusilazole (Flu) and epoxiconazole (Epo)) were assessed in Saccharomyces cerevisiae (yeast) via functional genome-wide knockout mutant profiling. A total of 169 (4.49%), 176 (4.67%), 198 (5.26%), 218 (5.79%) and 173 (4.59%) responsive genes were identified at three concentrations (IC50, IC20 and IC10) of Pro, Pen, Teb, Flu and Epo, respectively. The five conazoles tended to have similar gene mutant fingerprints and toxicity mechanisms. "Ribosome" (sce03010) and "cytoplasmic translation" (GO: 0002181) were the common KEGG pathway and GO biological process term by gene set enrichment analysis of the responsive genes, which suggested that conazoles influenced protein synthesis. Conazoles also affected fatty acids synthesis because "biosynthesis of unsaturated fatty acids" pathway was among the top-ranked KEGG pathways. Moreover, two genes, YGR037C (acyl-CoA-binding protein) and YCR034W (fatty acid elongase), were key fingerprints of conazoles because they played vital roles in conazole-induced toxicity. Overall, the fingerprints derived from the yeast functional genomic screening provide an alternative approach to elucidate the molecular mechanisms of environmental pollutant conazoles.

Lancefield Whole Blood Killing Assay to Evaluate Vaccine Efficacy.

While the Lancefield whole blood killing assay is named after the renowned streptococcal researcher Rebecca Lancefield, the protocol was first described by Todd in 1927 (Br J Exp Pathol 8:1-5, 1927). Initially, the assay was used to identify novel Group A Streptococcal (GAS) serotypes through the supplementation of non-immune human blood (often from infants) with type-specific antisera prepared in rabbits (Lancefield, J Exp Med 106:525-544, 1957; Maxted, Br J Exp Pathol 37:415-422, 1956) and to demonstrate the impressive longevity of type-specific immunity in patients following invasive GAS infection (Lancefield, J Exp Med 110:271-292, 1959). The modern assay is routinely used to screen defined GAS mutants (Wessels, Bronze, Proc Natl Acad Sci U S A 91:12238-12242, 1994; Zinkernagel et al., Cell Host Microbe 4:170-178, 2008) or transposon libraries (Le Breton et al., Infect Immun 81:862-875, 2013) for enhanced susceptibility to opsonophagocytic killing or to screen vaccine antisera (Salehi et al., mSphere 3:e00617-e00618, 2018) or other serological preparations (Reglinski et al., Sci Rep 5:15825, 2015) for anti-streptococcal activity.

In silico Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae.

Salmonella enterica is one of the most common pathogens associated with produce outbreaks worldwide; nonetheless, the mechanisms uncovering their interaction with plants are elusive. Previous reports demonstrate that S. enterica ser. Typhimurium (STm), similar to the phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000, triggers a transient stomatal closure suggesting its ability to overcome this plant defense and colonize the leaf apoplast. In order to discover new molecular players that function in the stomatal reopening by STm and Pst DC3000, we performed an Arabidopsis mutant screening using thermal imaging. Further stomatal bioassay confirmed that the mutant plants exo70h4-3, sce1-3, bbe8, stp1, and lsu2 have smaller stomatal aperture widths than the wild type Col-0 in response to STm 14028s. The mutants bbe8, stp1 and lsu2 have impaired stomatal movement in response to Pst DC3000. These findings indicate that EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria. The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens.

Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed "annealing at critical temperature PCR" (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.