Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants

Huffel, Van; Stock, Michiel; Ruttink, Tom; Baets, Bernard De

doi:10.3389/fpls.2022.907095

Cited by 9 publications

(9 citation statements)

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“…Several experimental design strategies have been proposed for multiplex CRISPR screens in plants using combinatorial gRNA libraries. These strategies aim to maximize coverage of relevant genetic interactions while minimizing the number of plants ( Van Huffel et al, 2022 ). Our use of GEDH can be considered as variations of these strategies in which first higher- order mutants are generated and subsequently deconvoluted to identify causative genotypes for the phenotypes of interest ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Combining multiplex gene editing and doubled haploid technology in maize

Impens,

Lorenzo,

Vandeputte

et al. 2023

New Phytologist

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A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain heteroallelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping.We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling.By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination.We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

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Section: Discussionmentioning

confidence: 99%

Combining multiplex gene editing and doubled haploid technology in maize

Impens,

Lorenzo,

Vandeputte

et al. 2023

New Phytologist

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“…SMAP design overcomes these limitations by designing highly specific amplicons at gRNA target sites for any number of user-selected genes and presents the user with compatible, ready-to-order designs. SMAP design greatly simplifies larger CRISPR experiments (e.g., multiplex and combinatorial CRISPR screens (37)) by reducing the design step from weeks or months to just hours of CPU time. The pre-designed genome-wide amplicons presented here effectively eliminate the design step altogether as users just need to select their gene identifier in the list and order the associated primers and/or gRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…SMAP design greatly simplifies larger CRISPR experiments (e.g. multiplex and combinatorial CRISPR screens ( 41 )) by reducing the design step from weeks or months to just hours of CPU time. The pre-designed genome-wide amplicons presented here effectively eliminate the design step altogether as users just need to select their gene identifier in the list and order the associated primers and/or gRNAs.…”

Section: Discussionmentioning

confidence: 99%

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

Develtere

Waegneer

Debray

et al. 2023

Nucleic Acids Research

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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.

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“…An increase in editing efficiency is particularly important when performing CRISPR screens to keep the mutant population small 34, 35, 53 . For example, when attempting to capture all 190 double mutants in a pool of 20 genes, a CRISPR vector with two gRNAs per vector and an overall mutation efficiency of 31% (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…For example, when attempting to capture all 190 double mutants in a pool of 20 genes, a CRISPR vector with two gRNAs per vector and an overall mutation efficiency of 31% (e.g. RPS5A::Cas9-SV40-P2A-mCherry-N7) will require an estimated population size of ~18,000 individuals 53 . A vector that confers 73% global mutation efficiency (e.g.…”

Section: Discussionmentioning

confidence: 99%

Continual improvement of multiplex mutagenesis in Arabidopsis

Develtere,

Decaestecker,

Rombaut

et al. 2023

Preprint

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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 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 and can still be optimized. We genotyped more than 6,000 Arabidopsis T2 plants to test seven promoters and eleven NLS architectures across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We find that the RPS5A promoter and double-BP NLS architecture were individually the most efficient components. When combined, 99% of T2 plant contained at least one knockout mutation and 84% contained 4-7-plex knock-outs. These optimizations will be useful to generate higher-order knockouts in the germline of Arabidopsis and likely be applicable to other CRISPR systems as well.

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Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants

Cited by 9 publications

References 50 publications

Combining multiplex gene editing and doubled haploid technology in maize

Combining multiplex gene editing and doubled haploid technology in maize

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

Continual improvement of multiplex mutagenesis in Arabidopsis

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