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Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin–antidote gene drive that impairs pollen germination

Abstract

Synthetic gene drives, inspired by natural selfish genetic elements and transmitted to progeny at super-Mendelian (>50%) frequencies, present transformative potential for disseminating traits that benefit humans throughout wild populations, even facing potential fitness costs. Here we constructed a gene drive system in plants called CRISPR-Assisted Inheritance utilizing NPG1 (CAIN), which uses a toxin–antidote mechanism in the male germline to override Mendelian inheritance. Specifically, a guide RNA–Cas9 cassette targets the essential No Pollen Germination 1 (NPG1) gene, serving as the toxin to block pollen germination. A recoded, CRISPR-resistant copy of NPG1 serves as the antidote, providing rescue only in pollen cells that carry the drive. To limit potential consequences of inadvertent release, we used self-pollinating Arabidopsis thaliana as a model. The drive demonstrated a robust 88–99% transmission rate over two successive generations, producing minimal resistance alleles that are unlikely to inhibit drive spread. Our study provides a strong basis for rapid genetic modification or suppression of outcrossing plant populations.

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Fig. 1: The CAIN gene drive design and predicted genetic behaviour.
Fig. 2: Transmission rate of CAIN from the T1 to F1 generation.
Fig. 3: Genotypes at the NPG1 locus for FAST+ F1 plants generated from male T1 plants carrying TPD-CAIN.
Fig. 4: Transmission rate of TPD-CAIN from the F1 to the F2 generation in reciprocal crosses.
Fig. 5: Genotypes at the NPG1 locus for FAST− F1 and FAST+/− F2 plants.
Fig. 6: Simulated spread dynamics of modification and suppression CAIN drives.

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Data availability

The Illumina sequencing data have been deposited in the Genome Sequence Archive58 in National Genomics Data Center59, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences under accession number CRA011573. Plasmid sequences have been deposited in GenBase (https://ngdc.cncb.ac.cn/genbase/?lang=en, accession ID: C_AA031173.1, C_AA031174.1 and C_AA031175.1).

Code availability

The scripts developed for variant calling at gRNA target sites and simulation of the population dynamics for CAIN are available at GitHub (https://github.com/QianLabWebsite/GeneDrive).

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Acknowledgements

This work was supported by grants from the Strategic Priority Research Program (Precision Seed Design and Breeding, grant no. XDA24020103, to W.Q.) and Project for Young Scientists in Basic Research (grant no. YSBR-078, to W.Q.) from the Chinese Academy of Sciences. We thank J. Zhang from University of Michigan, P. Thomas from University of Adelaide and C. Gao, T. Zhao and Y. Chen from Institute of Genetics and Developmental Biology Chinese Academy of Sciences for discussion. We thank X. Cao from Institute of Genetics and Developmental Biology Chinese Academy of Sciences for providing plasmid XF675.

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Contributions

W.Q. and Y.L. designed the study. Y.L. performed the experiments. B.J. and Y.L. performed the computational analyses. Y.L., J.C. and W.Q. wrote the manuscript.

Corresponding author

Correspondence to Wenfeng Qian.

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Competing interests

Y.L., B.J. and W.Q. have been granted a China invention patent (ZL202311247476.0) based on some results reported in this paper. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Incorporation of four gRNAs into CAIN.

a, Four gRNAs selected to be multiplexed into the CAIN constructs. Mutated nucleotides in the recoded version of NPG1, based on synonymous codons, are indicated by red squares. b, The structure of recoded NPG1. c, The position of four gRNA target sites on genomic NPG1 allele. Amplification of genomic regions containing the four target sites was performed using the NPG-gDNA-F1 and NPG-gDNA-R1_2 primers (in red). Sanger sequencing of the target sites was performed using four primers (NPG-gDNA-F1, NPG-gDNA-F2, NPG-gDNA-R1_1, and NPG-gDNA-R2, in purple).

Extended Data Fig. 2 Schematic maps of FAST only control construct and CAIN gene drives.

Shown are schematic maps of the FAST only control construct (a), DMC-CAIN (b), and TPD-CAIN (c) gene drive constructs, illustrating their total length and main features.

Extended Data Fig. 3 Experimental procedures overview.

a, Step 1: Introduction of control construct (FAST only), DMC-CAIN, or TPD-CAIN gene drive construct into Arabidopsis Col-0 plants, indicated by red fluorescence marker (FAST + ) in dry seeds. Single-locus insertion screening was performed using TAIL-PCR and whole genome resequencing. b, Step 2: Screened T1 plants were used as the male parent to cross with wild-type female parent. Transmission rate of the CAIN gene drive (CAIN%) was calculated from the fraction of F1 seeds exhibiting FAST marker. c, Step 3: Cultivation of F1 seeds to obtain F1 plants and genotyping (as described in Methods). d, Step 4: Crosses between F1 plants of known genotypes (male or female parent) and wild-type plants, calculating CAIN% in F2 seeds. e, Step 5: Cultivation of F2 seeds to obtain F2 plants and genotyping.

Extended Data Fig. 4 Mutation types detected in FAST+ F1 plants.

This figure illustrates the locations of specific bases involved in insertions, deletions, and single nucleotide polymorphisms generated at the gRNA2 (a) and gRNA11 (b) target sites. * denotes one potential alignment result, as the underlined bases can be positioned on either side of the deletion.

Extended Data Fig. 5 Transmission rate of DMC-CAIN from the F1 to F2 generation.

a, Diagram of genotyping in F1 plants’ inflorescence. b, Sanger sequencing-based genotyping results for 12 FAST + F1 plants at four gRNA target sites. c, Bar plots illustrating the mean DMC-CAIN transmission rate estimated from individual siliques.

Extended Data Fig. 6 Estimation of male germline cleavage efficiency, incomplete penetrance, and female germline cleavage efficiency of the TPD-CAIN system.

a, Estimated male germline cleavage efficiency and penetrance rate in F1 and F2 progeny. Among the F1 progeny, 94.3% (526/558, Supplementary Table 3) of the plants were FAST+ (TPD-CAIN/+), with all of them showing an NPG1 genotype at the gRNA11 target site (Supplementary Table 4). A small percentage, 2.6% (5.7% × 5/11), were +/+; NPG1+/− plants, and another 3.1% (5.7% × 6/11) were +/+; NPG1+/+ plants. When examining our F2 progeny, 94.8% (3868 out of 4080, Supplementary Table 3) were FAST+ (TPD-CAIN/+), all of which showed an NPG1 genotype at the gRNA11 target site (Supplementary Table 5). The remaining 5.2% F2 plants were all +/+; NPG1+/− (Supplementary Table 5). From these results, we estimated an average failed cleavage rate of 1.6%, which corresponds to a male germline cleavage efficiency of 98.4%. Additionally, we estimated an average penetrance rate of 96.0%. b, Estimation of female germline cleavage efficiency (r). We calculated the cleavage efficiency r using the genotypes of the NPG1 locus at the gRNA11 target site in FAST− F2 plants, assuming no further cleavage occurs in these plants. From an observed fraction of 1/34 +/+; NPG1+/+ plants (Supplementary Table 5), we estimated r to be 94.1%.

Extended Data Fig. 7 Potential application and confinement of the CAIN system.

a, Weed management application: inserting the CAIN system into a herbicide resistance gene of target weeds, and combining this with localized herbicide applications in the field, enables CAIN to facilitate spatially confined weed suppression. b, Confinement of CAIN spread via tethering with TARE. The TARE drive (or a similar variant) is confinable based on the initial release frequency of drive-carriers into the population (upper panel), in contrast to the CAIN system, which lacks such confinable characteristics (middle panel). To address its unconfinable nature, the CAIN (without Cas9 included in the drive construct), when tethered to TARE, exhibits functionality contingent upon the presence of Cas9 provided by TARE. This arrangement effectively confines the spread of the CAIN system (bottom panel).

Extended Data Fig. 8 Simulation analysis of factors influencing the spread dynamics of the CAIN system.

a-c, Computational simulations depicting the impact of inbreeding (a), fitness cost (b), and the fertility of male heterozygous CAIN carriers (c) on the spread dynamics of the CAIN system. d, Computational simulation depicting the combined effect of inbreeding and fitness cost on the spread dynamics of the CAIN system. e, Computational simulation depicting the spread dynamics of the CAIN system, both linked and unlinked with its target gene. f, The spread dynamics of the CAIN system inserted into a female fertility gene. g, The spread dynamics of the CAIN system inserted into a viability gene.

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Liu, Y., Jiao, B., Champer, J. et al. Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin–antidote gene drive that impairs pollen germination. Nat. Plants 10, 910–922 (2024). https://doi.org/10.1038/s41477-024-01692-1

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