Safeguarding CRISPR-Cas9 gene drives in yeast

Abstract

RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations could be used to address environmental and public health problems. However, the possibility of unintended genome editing occurring through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeast Saccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.

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Figure 1: Mechanism and population-level effect of endonuclease gene drives.
Figure 2: Biased inheritance of an ADE2 gene drive element in S. cerevisiae.
Figure 3: Gene drives and cargo genes remain intact upon copying and can spread by targeting both nonessential and essential genes.
Figure 4: Quantitative PCR shows relative abundance of wild-type and drive-containing alleles in diploids.
Figure 5: Available safeguards include targeting synthetic sequences and reversing drive-spread phenotypic changes with subsequent drives.

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Acknowledgements

We are very grateful to S. Doris, D. Spatt and F. Winston for their incredible patience, generosity and expertise in tetrad dissection. We also thank F. Winston for providing us with SK1 strains and members of the Church laboratory for insightful discussions. This work was supported by grants from the Department of Energy (DOE) (DE-FG02-02ER63445 to G.M.C.), National Science Foundation (NSF) (SynBERC SA5283-11210 and MCB-1330914 to G.M.C.), National Cancer Institute (NCI) (5T32CA009216-34 to A.C.), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (1K99DK102669-01 to K.M.E.) and the Wyss Institute for Biologically Inspired Engineering (Technology Development Fellowship to K.M.E.).

Author information

S.L.D. initiated the study; J.E.D., A.C., S.L.D. and K.M.E. designed the experiments; J.E.D. performed the experiments with assistance from A.C.; J.E.D., A.C., S.L.D. and K.M.E. analyzed the data; and K.M.E. wrote the paper with A.C. and contributing input from J.E.D., S.L.D. and G.M.C.

Correspondence to Kevin M Esvelt or George M Church.

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Competing interests

K.M.E. and G.M.C. are authors of a patent filed on CRISPR gene drive (PCT/US2015/010550). K.M.E. is author of a provisional patent filed on CRISPR gene drive (serial no. 62/236,545).

Integrated supplementary information

Supplementary Figure 1 Molecular confinement via 'split drive' sgRNA-only cassettes with chromosomal or episomal Cas9.

A) In transgenic laboratory populations expressing Cas9 (brown) from an unlinked locus such as another chromosome, the sgRNA-only drive (green) will be copied in every generation. For clarity, copying is assumed to occur when haploid cells combine to form a diploid. In our S. cerevisiae experiments, Cas9 was encoded on an episomal plasmid with imperfect inheritance that should produce a similar pattern. (B) If escaped organisms encoding an sgRNA-only drive mate with wild-type organisms, the cas9 gene quickly segregates away from the sgRNA-only drive, precluding exponential spread. Any organisms that do encode Cas9 will still exhibit drive, but the total number of copies is limited by the number of escaped organisms and therefore is dwarfed by the wild-type population. If one organism is released from the laboratory for every million wild-type organisms in the population, a perfectly efficient drive with no fitness cost will linearly increase in relative abundance by 2E-6 per generation. This tiny inheritance advantage is exceedingly unlikely to counterbalance the fitness cost of an actual split gene drive. (C) The episomal Cas9-expressing plasmid is unstable in the absence of active selection. With an average loss rate of ~3.8% per generation, more than 2/3 of yeast have lost the plasmid after a single round of asexual overnight growth (10 generations). While variable across independent mating experiments, the plasmid is typically lost at a rate of ~50% during meiotic sporulation, approximately equivalent to a chromosomal transgene. These high loss rates suggest there is minimal risk of Cas9 remaining available to bias the inheritance of the sgRNA-only cassette over generations. Indeed, mitotic loss suggests that the plasmid-encoded gene would likely be eliminated from the population more quickly than a chromosomally-integrated equivalent in the event of an accidental release.

Supplementary Figure 2 Reversal of drive-induced ADE2 loss by an overwriting drive.

Haploid yeast containing a complete autonomous ADE2-disrupting gene drive were mated with haploids containing an overwriting drive that restores ADE2 function. 15 diploid offspring were sporulated, dissected, and plated on adenine-limited plates. The resulting cream-colored colonies indicate that an intact ADE2 gene is present in all progeny, indicative of the ADE2-restoring drive successfully cutting and replacing the ADE2-disrupting gene drive.

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DiCarlo, J., Chavez, A., Dietz, S. et al. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat Biotechnol 33, 1250–1255 (2015). https://doi.org/10.1038/nbt.3412

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