Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.

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.

SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.

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.

BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize.

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.

A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants.

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.

The heat is on: a simple method to increase genome editing efficiency in plants.

BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

CRISPR screens in plants: approaches, guidelines, and future prospects.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools, and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene functions in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology.

CRISPR Screens in Plants: Approaches, Guidelines, and Future Prospects.

CRISPR-Cas systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in new powerful screens to test gene functions at the genomic scale. While there is tremendous potential for CRISPR screens to map and interrogate gene regulatory networks at unprecedented speed and scale, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports using this strategy to generate mutant knockout collections or diversify DNA sequences. In addition, we provide insight on how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene function in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the large resource of genomic profiles that were generated in the last two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will greatly advance plant functional and synthetic biology.