Daisy-chain gene drives for the alteration of local populations

Abstract

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. "Daisy-drive" systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

Keywords: CRISPR; ecological engineering; evolutionary dynamics; evolutionary genetics; gene drive.

Fig. 1.

Comparison of self-propagating and daisy-chain gene drive. (A) Self-propagating CRISPR gene drives distort inheritance in a self-propagating manner by converting wild-type (W) alleles to drive alleles in heterozygous germline cells. (B) A daisy-drive system consists of a linear chain of serially dependent, unlinked drive elements; in this example, A, B, and C are on separate chromosomes. Elements at the base of the chain cannot drive and are successively lost over time via natural selection, limiting overall spread. (C) Family tree resulting from the release of a single daisy-drive organism in a resident wild-type population in the absence of selection. On the right is a graphical depiction of the total number of alleles per generation. Throughout, chromosome illustrations represent genotypes in germline cells.

Fig. 2.

Dynamics of CBA daisy-chain gene-drive systems. (A) After being cut by an upstream daisy allele, a wild-type allele is repaired either by homologous recombination (HR), creating a second copy of the other allele at the locus, or by nonhomologous repair (e.g., NHEJ), leading to generation of a resistant allele. This process occurs in the germline and is independent at each locus. We assume that resistance at the cargo locus, A, is dominant lethal if inherited. (B) A highly efficient daisy drive ($95\%$ homing efficiency) with an $8\%$ fitness cost for the cargo element seeded at $2\%$ spreads the cargo nearly to fixation (Left). (B, Center) A low-efficiency drive ($60\%$) with the same initial release size no longer allows drive spread. (B, Right) Increasing the release size of the inefficient drive to $15\%$ again allows cargo spread to near fixation. (C) The maximum (Max) frequency achieved by cargo alleles as a function of the homing efficiency and the cargo fitness cost, for release sizes of $1\%$ (Left), $5\%$ (Center), and $10\%$ (Right). Throughout, we assume a $0.01\%$ fitness cost for C and B elements and neutral resistant alleles at the C and B loci.

Fig. 3.

Quantitative evaluation of cargo spread in a single population, for single and repeated releases. (A) Results assuming a single release of daisy-drive organisms in a wild population. (A, Left) Representative simulations assuming a $1\%$ release. (A, Right) Time to achieve $99\%$ frequency for varying release frequency. (B) Results assuming a constant rate of release of daisy-drive organisms. (B, Left) Representative simulations, assuming an initial 1% release with a subsequent release rate of 0.01 per generation (see SI Appendix, section 2.3 for details). (B, Right) Time to 99% frequency with varying release rate, which we set as both the initial release frequency as well as the subsequent continuous release frequency, indicated by the horizontal axis. (See SI Appendix, section 2.3 for details on continuous release.) All simulations assume a $10\%$ cargo cost, $0.01\%$ cost per upstream element, and $60\%$ (A and B, Top), $80\%$ (A and B, Middle), or $95\%$ (A and B, Bottom) homing efficiencies.

Fig. 4.

Modeling daisy-drive containment in a system of populations connected by gene flow. (Left) Illustration of the population structure: Five populations with equal sizes are connected in a chain, and each neighboring pair has bidirectional gene flow with rate $10^{-2}$ in each direction. The three columns in Right then correspond to the three scenarios described in the text: CBA daisy-chain drive (first column), self-propagating (“standard”) drive with multiple gRNAs targeting an essential gene, as in ref. (second column), and nondrive inundative release (third column). Frequencies over time are indicated for each allele in each of the populations. Drive-based simulations (daisy chain and standard) assume $80\%$ homing efficiency, $10\%$ dominant cargo element fitness cost, and $15\%$ release frequency. Daisy-chain drive simulations further assume $0.01\%$ upstream element (C, B) fitness cost. Inundative release simulations assume $10\%$ dominant fitness cost and $99.9\%$ release. See SI Appendix, section 5 for details.

Fig. 5.

Preventing the formation of “daisy necklaces.” (A) Any recombination event that moves a DNA template for guide RNA from one element to another, where it will be reliably copied, could create a daisy necklace capable of self-propagating drive. (B) Because promoters can be changed, repetition of the conserved DNA template sequence is a key problem. (C) Using existing data, we generated a template identifying candidate positions presumed tolerant of sequence changes. (D) Relative activities of candidate guide RNAs generated from the template were assayed by using a Cas9 transcriptional activator screen using a tdTomato reporter in human cells.