Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Abstract

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

Keywords: CRISPR; Drosophila; ERACR; MCR; active genetics; drive-neutralizing; e-CHACR; gene drive; modeling; risk management.

Figure 1:. Gene-drive and neutralizing drive elements

A) Scheme depicting gene-drives and neutralizing elements. Left: MCR (mutagenic chain reaction) gene-drive element carrying Cas9, an eGFP fluorescent marker, and a gRNA- for copying. Center: eCHACR carrying two or more gRNAs: one gRNA (blue) for copying at its genomic insertion site, a second gRNA (purple) targeting Cas9, and a DsRed (or eGFP) fluorescent eye marker. e-CHACRs are typically inserted at a different chromosomal site (locus A) than the gene-drive (locus B). Right: ERACR carrying gRNAs that target sequences flanking the MCR-GFP element and a DsRed marker. B) Two MCR elements inserted at the same site in the y locus: 1) MCR lacking a fluorescent marker (third row), and 2) MCR-GFP, an eGFP-marked version (bottom row) carrying the same core components (vasa-Cas9 and gRNA-y1). C) Cross scheme for generating MCR-GFP F₁ “master females” and their F₂ progeny. Phenotypes of F₀, F₁, and F₂ progeny are depicted schematically. D) Percentage of GFP⁺ F₂ progeny (carrying MCR-GFP element) recovered per cross. Fly heads depict eye phenotypes determined by the white (w) locus: wild-type = w⁺ (red eyes), recessive w⁻ (white eyes), eye fluorescence markers (GFP = radiating green lines), and body color (y⁺ = brown, y⁻ = yellow).

Figure 2:. e-CHACR-wR versus the MCR-GFP element

A) Schematic of DsRed⁺ e-CHACR-wR element linked to the ac⁴ allele (e-wR^ac) and the y⁺ ac⁺ marked MCR-GFP allele. B) Crossing scheme for testing the e-CHACR-wR (e-wR) against the MCR-GFP element. e-CHACR-wR females were mated to MCR-eGFP males to generate F₁ master females that were then pair-mated to ac⁴ (ac⁻) males. F₂ progeny were screened and analyzed for MCR-GFP presence and presence (DsRed⁺) or absence (w^NHEJ) of e-CHACR-wR on ac⁺ or ac⁴ marked chromosomes. C,C’) Percentage of fluorescence phenotypes in total female (C) or male (C’) F₂ progeny per cross. D,D’) Prevalence of GFP⁺ and GFP⁻ alleles in MCR-GFP donor (left) and receiver F₂ D) females or D’) males (right). E,E’) Prevalence of body color in total F₂ E) females or E’) males. F) Percentage of MCR-GFP donor (ac⁺, black dots) and receiver (ac⁻, pink dots) alleles in F₂ males.

Figure 3:. ERACR construct designs and crossing schemes.

A) Diagrams of MCR-GFP and ERACR elements. Fly heads on the left indicate the phenotype of each strain. Schematic not drawn to scale. B) Cross schemes to generate F₁ “master females” with ERACRs in-trans to the MCR-GFP element and their F₂ progeny. Crossing schemes for y⁻ ERACR-min (left) and for y⁺ ERACR-1 and y⁺ ERACR-2 (right). Fly heads depict eye color (wild-type = w⁺ (red), w⁻ (white), or w^a (orange), eye fluorescence (radial emanating lines of eGFP (green), DsRed (red), both eGFP and DsRed (alternating green and red), or neither fluorescence (white), and body color: y⁺ (brown) or y⁻ (yellow). Expected F₂ phenotypes bolded and outlined with black boxes.

Figure 4:. ERACRs delete and replace the MCR-GFP drive

Phenotypic frequencies and deduced gene conversion events in F₂ progeny from crosses depicted in Fig. 3A. (additional crossing schemes in Figs. S13A–E). Black type = mean of percentages across all vials and orange type = estimated receiver conversion frequencies (e.g., DsRed w^a males/total w^a males) (see Data S3 for details). A) DsRed⁺ inheritance is a proxy for scoring ERACR prevalence (DsRed⁺, GFP⁻ and DsRed⁺, GFP⁺ progeny included). A’) The subset of data plotted in panel 4A with traceable donor and receiver chromosomes re-plotted by donor (w⁻; left graph) versus receiver (w^a; right graph) chromosomes. B) Proportion of F₂ males or females inheriting the donor (w⁻) versus receiver (w^a) chromosome. C,E) Schematics illustrating predicted gene conversion events responsible for specific phenotypes: sequences of relevant junctions shown in (Fig. S14). D) GFP inheritance is a proxy for MCR-GFP prevalence (DsRed⁺,GFP⁺ plus DsRed⁻,GFP⁺ F₂ progeny). E) MCR-GFP alleles, although intact, have NHEJ-induced indels at the y2 and y3 cut sites.

Figure 5:. Alternative ERACR versus MCR-GFP outcomes

Partial copying or fusion of sequences on the receiver chromosome. A) Non-fluorescent DsRed⁻,GFP⁻ F₂ progeny as a proxy for MCR-GFP deletion events. A’) Data in panel A re-plotted by inheritance of y⁻ (lethal deletion) versus y⁺ (recombination) alleles. Body color permits inference of the type of excision event that occurred, as depicted in C-E. B) Loss of essential sequences distal to the ERACR gRNA cut sites result in male-lethal alleles. Viability of several such alleles can be rescued in males by duplications covering the tip of the X chromosome (Fig. S12B). C-E) The MCR-GFP element is deleted, but not replaced by ERACR, producing three distinct observed outcomes. C) Hypothesized repair mediated by partial pairing of un-recoded y sequences carried by ERACR-1 and endogenous y sequences 3’ to the MCR-GFP element result in expression of the recoded y cassette and a wild-type body color. D) NHEJ events joining adjacent sequences at the gRNA-y2 and gRNA-y3 cut sites. E) Likely pairing between 17 bp of the gRNA-y2 genomic target sequences 5’ to the MCR-GFP with correspondingly oriented sequences in the gRNA-y2 transgene carried by either ERACR-min or ERACR-1. F) Prevalence of DsRed⁺, GFP⁺ F₂ progeny in which MCR-GFP and ERACR sequences are both present on the receiver chromosome. G) Two examples of MCR-GFP/ERACR fusion events (see Fig. S14).

Figure 6:. Drive performance of single-cut ERACRs versus MCR-GFP drive

A) Schemes illustrating single-cut and double-cut ERACR designs. B) Crossing scheme for generating and testing transmission by single-cut w^a ERACR/MCR-GFP F₁ master females. C-E) Single-cut versions of ERACR-2 are placed in-trans to the MCR-GFP element where the gRNA-y2 and gRNA-y3 are separated by a distance of 11.3 kb (see also Fig. S19 for Copy-Cat analysis). C) Fluorescent phenotypes for single-cut DsRed⁺ ERACR2-y2 and ERACR2-y3 and MCR-GFP. D) Percent DsRed⁺ females or males inheriting either the donor (w⁻) ERACR-2 single-cut chromosome or the receiver (w^a) chromosome. E) Donor versus receiver chromosome transmission for single-cut ERACR2-y2 and ERACR2-y3.

Figure 7:. Cage experiments: MCR-GFP versus e-CHACR-wR and ERACRs

A,C,E) Modeling of MCR-GFP versus e-CHACR-wR (A) and ERACR (C,D) dynamics (solid lines: same as B,D,E) and model fits (dotted lines) plotted for the frequencies of MCR-GFP (green) and e-CHACR-wR or ERACRs (red). B) Plot of fraction of individuals with different phenotypes over 12 generations. Green = MCR-GFP and Red = e-CHACR-wR prevalence in the total population (e.g., both males and females). Orange = females carrying both elements. Yellow = females with mosaic eyes indicative of Cas9 activity. Dark traces represent separate cage replicates and pale lines = model simulations (also in panels D,F). C,E) Modeling of MCR-GFP versus ERACR dynamics over 26 generations (C) and ERACR-2 (E). D,F) ERACR-min (D) and ERACR-2 (F) versus MCR-GFP cage experiments. Red = DsRed⁺ ERACRs. Green = MCR-GFP⁺. Yellow = both markers.

Figure 8:. Summary Diagrams

A) Diagram illustrating the generic action of the e-CHACRs against the MCR-GFP gene-drive. B) Diagram outlining the structures and homologous sequences serving as sites for potential SDSA-mediated partial copying between ERACR-2 and the MCR-GFP constructs (lightly shaded parallelograms). y1*, y2* and y3* indicate the loss of the gRNA-y1, gRNA-y2 and gRNA-y3 cut sites accompanying genomic insertion of the MCR-GFP element (gRNA-y1) or ERACRs (gRNA-y2 and gRNA-y3). C) Pie charts summarizing key e-CHACR performance parameters. D) Pie chart summary of F₂ male and female progeny outcomes derived from F₁ ERACR-2/MCR-GFP master females separated by inferred donor (w⁻ = red shaded sectors) versus receiver (w^a: peach = ERACR copied, green = MCR-GFP retention; gray =MCR-GFP deleted) chromosomes.