Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

Bollier, Norbert; Buono, Rafael Andrade; Jacobs, Thomas B.

doi:10.1101/2020.11.13.381046

Cited by 6 publications

(5 citation statements)

References 23 publications

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“…It has been well documented that mutagenic activity differs between individual primary transformants, and that editing at a primary locus increases the likelihood of editing at further loci (e.g., Bollier et al , 2020; Li et al , 2020). Similarly, we detected mutant phenotypes at higher frequencies in families that were already scored as er gl1 double mutants in the T 1 generation (Table S2).…”

Section: Discussionmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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SUMMARY Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

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Section: Discussionmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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“…Paired end sequencing was performed with Eurofins NGSelect amplicons (5M reads 2×150bp). Reads were demultiplexed using Je-demultiplex [66] and individual fastq files were obtained as previously described [67] using a Galaxy workflow (https://usegalaxy.be). Demultiplexing statistics can be found in Additional file 3.…”

Section: Methodsmentioning

confidence: 99%

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

Gaillochet

Fernández

Goossens

et al. 2022

Preprint

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The ever-increasing number of CRISPR components creates a significant burden when developing new genome engineering tools. Plant biotechnology in particular has few high-throughput options to perform iterative design-build-test-learn cycles when creating new gene-editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle – from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors. Given that previous LbCas12a-ABEs have low or no activity in plants, we used ITER to develop an optimized LbCas12a-ABE. We show that the sequential improvement of five components –NLS, crRNA, LbCas12a, adenine deaminase and linker– led to a remarkable increase in ABE activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirmed the activity of LbCas12a-ABE at endogenous targets and in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and LbCas12a-CBE. 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.

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“…Paired-end sequencing was performed with Eurofins NGSelect amplicons (5M reads 2x150bp). Reads were demultiplexed using Je-demultiplex [72] and individual fastq files were obtained as previously described [73] using a Galaxy workflow (https:// usega laxy. be).…”

Section: Genotyping and Ngs Analysismentioning

confidence: 99%

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

et al. 2023

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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.

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Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

Cited by 6 publications

References 23 publications

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

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

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

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