Safeguarding CRISPR-Cas9 gene drives in yeast

Journal name:
Nature Biotechnology
Volume:
33,
Pages:
1250–1255
Year published:
DOI:
doi:10.1038/nbt.3412
Received
Accepted
Published online

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.

At a glance

Figures

  1. Mechanism and population-level effect of endonuclease gene drives.
    Figure 1: Mechanism and population-level effect of endonuclease gene drives.

    (a) Homing endonucleases cut competing alleles, inducing the cell to repair the damage by copying the endonuclease gene. (b) By converting heterozygous germline cells into homozygotes containing two copies (teal), gene drives increase the odds that they will be inherited and consequently spread themselves and associated changes through wild populations (gray). Reproduced from reference 1.

  2. Biased inheritance of an ADE2 gene drive element in S. cerevisiae.
    Figure 2: Biased inheritance of an ADE2 gene drive element in S. cerevisiae.

    (a) Mutations in ADE2 generate a red phenotype on adenine-limiting media due to the buildup of red pigments. Mating a red mutant haploid to a wild-type haploid produces cream-colored diploids, which yield 50% red and 50% cream-colored progeny upon sporulation. (b) When haploids with a gene drive element targeting ADE2 mate with wild-type haploids in the presence of Cas9, cutting and subsequent replacement or disruption of ADE2 produces red diploids that upon meiosis yield exclusively red progeny. (c) Diploids produced by mating wild-type and ade2::sgRNA gene drive haploids yield cream-colored colonies in the absence of Cas9 or when the target site is removed by recoding but uniformly red colonies when both are present, demonstrating Cas9-dependent disruption of the wild-type ADE2 copy. (d) Spores from 15 dissected tetrads produce uniformly red colonies on adenine-limited plates, confirming disruption of the ADE2 gene inherited from the wild-type parent. In the absence of the target site or Cas9, normal 2:2 segregation is observed.

  3. Gene drives and cargo genes remain intact upon copying and can spread by targeting both nonessential and essential genes.
    Figure 3: Gene drives and cargo genes remain intact upon copying and can spread by targeting both nonessential and essential genes.

    (a) The ADE2-targeting gene drive was modified to carry URA3 as a cargo gene. (b) Diploids produced by mating wild-type URA3 haploid yeast with haploids encoding the gene drive carrying URA3 were allowed to sporulate and tetrads dissected to isolate colonies arising from individual spores. Pictures are spores from 15 of these tetrads. All grew on replica plates lacking uracil, demonstrating that the drive successfully copied URA3 in all diploids. (c) A gene drive designed to cut and recode the 3′ end of the essential ABD1 gene.

  4. Quantitative PCR shows relative abundance of wild-type and drive-containing alleles in diploids.
    Figure 4: Quantitative PCR shows relative abundance of wild-type and drive-containing alleles in diploids.

    Highly efficient inheritance biasing by split drives across diverse yeast strains in the presence of Cas9 resulted from matings between SK1 haploids bearing gene drives and diverse wild-type haploid strains. “No Cas9” and “No target” refer to haploid cells containing the ADE2 drive mated to wild-type haploids in the absence of Cas9 or to an otherwise wild-type strain with Cas9 that has a mutation in the targeted sequence that blocks cutting. “2nd gen.” refers to the haploid progeny of an earlier mating. Data points are from independent cultures or mating events and represent the mean of three technical replicates.

  5. Available safeguards include targeting synthetic sequences and reversing drive-spread phenotypic changes with subsequent drives.
    Figure 5: Available safeguards include targeting synthetic sequences and reversing drive-spread phenotypic changes with subsequent drives.

    (a) An autonomous Cas9+sgRNA gene drive that cuts and replaces the recoded ADE2 gene. (b) Quantitative PCR results depicting the relative abundance of wild-type and drive-containing alleles in diploids arising from matings between SK1 haploids bearing the above gene drive and wild-type SK1 yeast. (c) A drive that cuts the autonomous drive and restores ADE2. (d) Quantitative PCR results for diploids arising from matings between SK1 haploids bearing the ADE2-disrupting and ADE2-restoring gene drives. Data points are from independent cultures (n = 3 technical replicates).

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

  7. Reversal of drive-induced ADE2 loss by an overwriting drive.
    Supplementary Fig. 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.

Accession codes

Primary accessions

NCBI Reference Sequence

References

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Author information

  1. These authors contributed equally to this work.

    • James E DiCarlo &
    • Alejandro Chavez

Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • James E DiCarlo,
    • Alejandro Chavez,
    • Sven L Dietz &
    • George M Church
  2. Harvard Medical School, Boston, Massachusetts, USA.

    • James E DiCarlo,
    • Alejandro Chavez,
    • Sven L Dietz,
    • Kevin M Esvelt &
    • George M Church
  3. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

    • James E DiCarlo
  4. Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.

    • Alejandro Chavez,
    • Sven L Dietz,
    • Kevin M Esvelt &
    • George M Church
  5. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Alejandro Chavez
  6. Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

    • Sven L Dietz

Contributions

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.

Competing financial 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).

Corresponding authors

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Molecular confinement via 'split drive' sgRNA-only cassettes with chromosomal or episomal Cas9. (262 KB)

    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.

  2. Supplementary Figure 2: Reversal of drive-induced ADE2 loss by an overwriting drive. (289 KB)

    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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    Supplementary Figures 1 and 2, Supplementary Tables 1 and 2 and Supplementary Note

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