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Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80–90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

CRISPR/Cas is now the standard technique to generate novel plant genotypes. However, optimizing the efficiency of the system continues to be an aspect of research and development. One of the improvements for increasing mutagenesis efficiency in different species is the application of heat stress. However, many experimental setups are limited by the requirement of using dedicated climate chambers to impose heat stress and by difficulties in the phenotyping of soil-grown plants. Here, we describe a simplified heat stress assay for in vitro-grown plants that can be completed in 6 days using commonly available laboratory equipment. We show that three 24-hr heat shocks (3×HS) at 37°C alternated with 24 hr of recovery at 21°C efficiently increases indel rates of LbCas12a and Cas9. We illustrate how visual mutant phenotypes (pds3 and gl1) can assist in quantifying genome editing efficiency, and describe how to quantify genome editing efficiency using genotyping by Sanger sequencing. We also provide a support protocol to efficiently clone a CRISPR expression vector in a single step. Together, our methods allow researchers to increase CRISPR-induced mutations using a low-tech setup in plants. © 2022 Wiley Periodicals LLC. Basic Protocol 1: 3×HS protocol Basic Protocol 2: Genotyping by Sanger sequencing Support Protocol: One-step cloning of a CRISPR expression vector.

Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design into a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the primer designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis and soybean. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

Background Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. Results We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle — from design to results — within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components — NLS, crRNA, LbCas12a, adenine deaminase, and linker — leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. Conclusion Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Though new technologies are being applied to tackle the problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in gene engineering via the CRISPR/Cas technology pave the way to accelerate plant breeding and meet this increasing demand. Here, we present a gene discovery pipeline named ‘BREEDIT’ that combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought resistance. We induced gene knockouts in 48 growth-related genes using CRISPR/Cas9 and generated a collection of over 1000 gene-edited maize plants. Edited populations displayed, on average, significant increases of 5 to 10% for leaf length and up to 20% for leaf width compared with controls. For each gene family, edits in subsets of genes could be associated with increased traits, allowing us to reduce the gene space needed to focus on for trait improvement. We propose BREEDIT as a gene discovery pipeline which can be rapidly applied to generate a diverse collection of mutants to identify subsets of promising candidates that could be later incorporated in breeding programs.

Otolaryngology involves the treatment of patients with diseases and disorders of the ear, nose, throat (ENT), and related structures of the head and neck. Many medical students in Canada have limited experiences in ENT and a vast majority of these students go on to pursue a career as primary care physicians. Physicians at a primary care facility classified patient's visits as either being "ENT" related or not, to assess the amount of ENT related concerns they typically encounter. The data was collected separately in the summer and winter months to assess any seasonal variability. One in eight patient encounters presented with an ENT related concern. The percentage of ENT related symptom presentation visits in the pediatric population for both data collection periods (29%) was more than three times that of the adult population (9%). The rate of ENT symptom presentation in both adult and pediatric populations was not affected by seasonality. Primary care physicians will encounter new patients presenting with ENT related concerns quite frequently. This is especially true in the pediatric patient population. Increased ENT medical education is both necessary and essential for undergraduate medical students, residents, and primary care physicians.L’oto-rhino-laryngologie (ORL) concerne les maladies et les troubles de l’oreille, du nez, de la gorge et des structures connexes de la tête et du cou. De nombreux étudiants au Canada n’ont qu’une expérience limitée de cette spécialité alors que la grande majorité d’entre eux poursuivent une carrière de médecin de soins primaires. Les médecins d’un établissement de soins primaires ont classé les visites des patients afin de déterminer le volume de consultations en lien avec l’ORL. Les données ont été recueillies séparément pendant les mois d’été et d’hiver pour évaluer la variabilité saisonnière. D’après les données, une consultation sur huit était liée à la présence de symptômes ORL. Le pourcentage de consultations chez la population pédiatrique pour les deux périodes de collecte de données (29 %) était plus de trois fois supérieur à celui de la population adulte (9 %). La survenance de symptômes ORL n’était pas affectée par la saisonnalité, ni chez l’une ni chez l’autre. Les médecins de soins primaires voient assez souvent de nouveaux patients présentant des problèmes ORL, particulièrement des enfants. Un renforcement de l’enseignement de la médecine ORL est à la fois nécessaire et essentiel pour les étudiants en médecine de premier cycle, les résidents et les médecins de soins primaires.

Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called “oligo stitching”. Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching’s versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist’s toolbox.Abstract Graphic

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

SUMMARYAdvanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Here we reveal, in the plant model Arabidopsis thaliana, that the PIN7 gene, which encodes a polarly localized transporter for the phytohormone auxin, produces two evolutionary-conserved transcripts. These isoforms PIN7a and PIN7b, differing in a 4 amino acid motif, are present at nearly equal levels in most cells. Although both variants do not differ in the subcellular localization and transport auxin with similar capacity, they closely associate and mutually influence their stability within the plasma membrane. Phenotypic complementation tests reveal that the functional contribution of PIN7b per se is minor but it markedly attenuates the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. These results establish alternative splicing of the PIN family as a conserved, functionally-relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.

The epidermal pavement cell shape in Arabidopsis is driven by chemical and mechanical cues that direct partitioning mechanisms required for the establishment of the lobe- and indentation-defining polar sites. Brassinosteroid (BR) hormones regulate pavement cell morphogenesis, but the underlying mechanism remains unclear. Here, we identified two PLECKSTRIN HOMOLOGY GTPase-ACTIVATING proteins (PHGAPs) as substrates of the GSK3-like kinase BR-INSENSITIVE2 (BIN2). The phgap1phgap2 mutant displayed severe epidermal cell shape phenotypes, and the PHGAPs were markedly enriched in the anticlinal face of the pavement cell indenting regions. BIN2 phosphorylation of PHGAPs was required for their stability and polarization. BIN2 inhibition activated ROP2-GTPase signaling specifically in the lobes because of PHGAP degradation, while the PHGAPs restrained ROP2 activity in the indentations. Hence, we connect BR and ROP2-GTPase signaling pathways via the regulation of PHGAPs and put forward the importance of spatiotemporal control of BR signaling for pavement cell interdigitation.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs. BREEDIT is a gene discovery pipeline that combines multiplex CRISPR/Cas9 genome editing of whole gene families with crossing schemes to improve complex quantitative traits.