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Cleave and Rescue gamete killers create conditions for gene drive in plants

Abstract

Gene drive elements promote the spread of linked traits and can be used to change the composition or fate of wild populations. Cleave and Rescue (ClvR) drive elements sit at a fixed chromosomal position and include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Here we demonstrate the essential features of the ClvR gene drive in the plant Arabidopsis thaliana through killing of gametes that fail to inherit a ClvR that targets the essential gene YKT61. Resistant alleles, which can slow or prevent drive, were not observed. Modelling shows plant ClvRs are robust to certain failure modes and can be used to rapidly drive population modification or suppression. Possible applications are discussed.

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Fig. 1: ClvR behaviour in a diploid plant and construct design.
Fig. 2: Genetic evidence for ClvR-based gamete killing and rescue.
Fig. 3: ClvR-based gamete killing and rescue is stable over multiple generations.
Fig. 4: Characterization of the target locus following exposure to ClvRubq7, and genetic behaviour of LOF mutations found in non-ClvR progeny of a female ClvR parent.
Fig. 5: Predicted behaviour of ClvR for population modification and suppression.
Fig. 6: ClvR gamete drive for population modification tolerates the presence of substantial frequencies of Rescue-only elements lacking Cas9 or gRNA function.
Fig. 7: ClvR gamete drive for population suppression tolerates the presence of modest frequencies of Rescue-only elements lacking Cas9 or gRNA function.
Fig. 8: Population modification but not population suppression can occur in the presence of resistant alleles.

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Data availability

All data are available in the main text and the Supplementary Information files. Illumina sequencing reads were deposited to SRA (bioproject, PRJNA1074841). The Arabidopsis TAIR 10 genome assembly was used in this study. Constructs and seeds of transgenic plants created in this study are available upon request.

Code availability

Modelling code and more information on the model, the scripts and parameters used to generate the data, and the data itself can be found at https://github.com/HayLab/Pigss. Plots were generated in R (version 4.2.3) with the ggplot2 package122.

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Acknowledgements

We thank E. Meyerowitz and members of the Meyerowitz Lab Paul Tarr and Carla de Agostini Verna for introducing us to techniques for Arabidopsis maintenance, transgenesis and crossing. This work was supported by a grant to B.A.H. from the Caltech Center for Evolutionary Science (G.O. and M.L.J.) and the Caltech Resnick Sustainability Institute Explorer Grant (G.O.). T.I. was supported by NIH Training grant number 5T32GM007616-39 and with support to B.A.H. from the US Department of Agriculture, National Institute of Food and Agriculture (NIFA) specialty crop initiative under US Department of Agriculture NIFA award number 2012-51181-20086.

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Contributions

Conceptualization, G.O., T.I. and B.A.H.; methodology, G.O., T.I., M.L.J., I.A. and B.A.H.; investigation, G.O., M.L.J., I.A. and B.A.H.; writing—original draft, G.O. and B.A.H.; writing—review and editing, G.O., T.I., M.L.J., I.A. and B.A.H.; funding acquisition, B.A.H.

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Correspondence to Bruce A. Hay.

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Competing interests

The authors have filed patent applications on ClvR and related technologies (US application numbers 15/970,728 and 16/673,823).

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Nature Plants thanks Meru Sadhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Alignment of the recoded A. lyrata rescue coding region to the A. thaliana target.

Guides are indicated as red arrows. Note that the full sequence of the A. lyrata YKT61 genomic region used for rescue (Supplementary File 1) contains many additional differences from the equivalent A. thaliana sequence, in regulatory sequences, introns and 5′ and 3′ UTR. The amino acid sequences of the two proteins are identical.

Extended Data Fig. 2 T3 heterozygous ClvR crosses for (a) female ClvRap and (b) male ClvRap.

T3 ClvRap/+ heterozygotes were grown to adulthood and their ovules (left three columns) or pollen (right three columns) used in outcrosses to WT. Bar graphs show the number of siliques scored (red circles) and the percent ClvR seeds produced in the T5 generation. The number of seeds within each silique scales with circle size. Counts are in Supplementary Table (ClvRap Crosses).

Extended Data Fig. 3 T3 heterozygous ClvR crosses for (a) female ClvRCaMV35S and (b) male ClvRCaMV35S.

T3 ClvRCaMV35S/+ heterozygotes were grown to adulthood and their ovules (left three columns) or pollen (right three columns) used in outcrosses to WT. Bar graphs show the number of siliques scored (red circles). The number of seeds within each silique scales with circle size. Counts are in Supplementary Table (ClvRCaMV Crosses).

Extended Data Fig. 4 Images of individual (a) and whole pots (b) of heterozygous ClvRubq, homozygous ClvRubq and WT plants.

In a individual plants have been removed from their pots and laid flat against a black background. b shows pots containing multiple plants.

Extended Data Fig. 5 Crossing scheme for (a) T3 and (b) T4 crosses discussed in text and Figs. 2 and 3.

(a) We selected 5 independent ClvRubq lines that showed 100% ClvR in the T2 self cross. Pollen of T2 plants was outcrossed to WT to generate T3 heterozyogtes. For each of these 5 independent lines we set up reciprocal crosses to WT with 4 plants per line (4 crosses/siliques per plant). (b) For 1 of the line from (A) ClvRubq7 we repeated the reciprocal crosses, with seeds coming from a ♀ClvR/+ or ♂ClvR/+ parent. For each of these we again crossed 4 plants (4 crosses/siliques per plant). Arabidopsis icons adapted from BioRender (2023), Structure of Arabidopsis thaliana.

Extended Data Fig. 6 Escaper genotyping.

(a-b) PCR amplifications of a 675 bp DNA fragment of the RFP marker for escapers from a ♀ClvR/+ X WT (a) or ♂ClvR/+ X WT (b) cross. Hetero- and homozygous (het, hom) ClvR plants were used as positive controls, WT as negative control. Only ClvR-bearing plants showed the RFP band. (c-d) Control PCRs on the same DNA samples as in a and b, in which the YKT61 target region was amplified. Note some female escapers in c had larger deletions. This experiment was carried out once.

Extended Data Fig. 7 Predicted behavior of ClvR for population modification and suppression.

(a-c) Population modification. ClvR is introduced as homozygous males at a frequency of 20% of the starting population, which is at carrying capacity, 10,000 individuals. The mating system is monogamous (a), or polyandrous, with 5 males each providing 1/5th of the pollen needed to fertilize all ovules of an individual female (b), or 20 males each providing 1/20th of the pollen needed (c). Fitness costs are incurred by gametes (a probability of not being able to participate in fertilization, if chosen by the model). Maternal carryover is set to zero. Lines represent the average of 10 runs. (d-f) Population suppression with a transgene inserted into a recessive locus required for female sporophyte fertility. ClvR is introduced as above, at a frequency of 20%. The mating system is monogamous (d), or polyandrous, with 5 males each providing 1/5th of the pollen needed to fertilize all ovules of an individual female (e), or 20 males each providing 1/20th of the pollen needed (f). Fitness costs are as above. Maternal carryover is set to zero or 30% (the approximate value observed in our experiments with ClvRubq). (g-i). As with d-f, but with the ClvR inserted into a locus required for male sporophyte fertility. For these simulations homozygous females were released into the population since homozygous males are sterile. Lines represent the average of 10 runs. For all panels compare with 10% introduction frequency data shown in Fig. 5.

Extended Data Fig. 8 Predicted behavior of ClvR for population suppression with 100% maternal carryover.

ClvR is introduced at a frequency of 10%, and is present in a female fertility locus (a-c) or a male fertility locus (d-f), thereby creating a LOF allele. (a-c) When ClvR is located in a gene required for female sporophyte fertility high levels of maternal carryover prevent population extinction. (d-f) In contrast, when ClvR is located in a gene required for male sporophyte fertility, population extinction is slowed but not prevented.

Extended Data Fig. 9 Effects of higher levels of elements lacking Cas9 on ClvR-mediated population suppression.

(a) ClvR, located inside a gene required in the sporophyte for female fertility, is introduced at a frequency of 10%, with 20% of these elements lacking Cas9. Individual runs are shown in thin lines and the average as a thick line. (b) ClvR, located in a gene required in the sporophyte for female fertility, is introduced at a frequency of 10%. Cas9 is located 1 map unit (1% recombination rate) away from the Rescue/gRNAs. Multiple individual runs fail to go to extinction while others that do go to extinction take much longer than under the conditions shown in Fig. 5.

Extended Data Fig. 10 Movement of a population suppression ClvR or a WT allele of the sporophyte fertility gene to a new unlinked location negatively affects population suppression.

(a-b) ClvR, located inside a gene required in the sporophyte for female fertility, is introduced at a frequency of 10%, with 1 × 10-6 of the ClvR elements having been transposed to a new unlinked locus, which is not required for female fertility. Individual runs are shown here, in a linear scale (a) and logarithmic scale (b). (c-d) ClvR, located inside a gene required in the sporophyte for female fertility, is introduced at a frequency of 10%. Additionally, a translocated WT version of the fertility locus is present at a third locus, not associated with ClvR, at an allele frequency of 5 × 10-7 (100 out of 100,000,000 individuals are heterozygous for this translocated fertility gene), such that individuals with that gene may be both ClvR homozygous and fertile. Individuals runs are shown on a linear scale (c) and a logarithmic scale (d).

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Table 1

Cross data and cleavage events.

Supplementary Data 1

Annotated plasmid maps with primers.

Supplementary Data 2

Sequencing files.

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Oberhofer, G., Johnson, M.L., Ivy, T. et al. Cleave and Rescue gamete killers create conditions for gene drive in plants. Nat. Plants 10, 936–953 (2024). https://doi.org/10.1038/s41477-024-01701-3

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