Repeat mediated excision of gene drive elements for restoring wild-type populations

Abstract

Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair. ReMEDE was incorporated into the mutagenic chain reaction (MCR) gene drive targeting the yellow gene of Drosophila melanogaster, successfully replacing drive alleles with wild-type alleles. Sequencing across the Cas9 target site confirmed transgene excision by SSA after pair-mated outcrosses with yReMEDE females, revealing ~4% inheritance of an engineered silent TcG marker sequence. However, phenotypically wild-type flies with alleles of indeterminate biogenesis also were observed, retaining the TGG sequence (~16%) or harboring a silent gGG mutation (~0.5%) at the PAM site. Additionally, ~14% of alleles in the F2 flies were intact or uncut paternally inherited alleles, indicating limited maternal deposition of Cas9 RNP. Although ReMEDE requires further research and development, the technology has some promising features as a gene drive mitigation strategy, notably its potential to restore wild-type populations without additional transgenic releases or large-scale environmental modifications.

Fig 1. Graphical summary.

Goldilocks scenario where a CRISPR/Cas9-based autonomous homing gene drive spreads through a target population over a defined period of time, after which the transgene is gradually removed through SSA restoring a wild-type phenotype.

Fig 2. Technology overview.

Schematic depicting homologous recombination (HR), end joining (EJ), and single strand annealing (SSA) in an autonomous homing gene drive containing the ReMEDE system.

Fig 3. Repeat Mediated Excision of a Transgene (ReMET).

(A) Schematic depicting ReMET constructs (in trans and in cis) and their removal by SSA. The yReMET construct (shaded in purple) contains EGFP and RFP under the control of independent 3xP3 promoters, and an endonuclease, I-SceI, under the control of UASp. The construct is flanked by a pair of direct repeats (DR) that are 30, 250, or 500 bp in length. Insertion of the ReMET construct into exon 2 of the yellow gene creates a null allele that produces yellow body pigmentation with both green and red eye fluorescence. I-SceI may be expressed by crossing with a nos-Gal4 driver line (in trans), or by expression from a RU486-inducible nos-GS (in cis). Expression of I-SceI generates a DSB at the recognition site (I-Site) located between the DRs, excising the transgene and one of the direct repeats through SSA. SSA-mediated removal of the transgene restores wild-type body pigmentation (brown body) with simultaneous loss of eye-specific fluorescence. The engineered synonymous mutation TGG->TcG provides molecular confirmation of ReMET. (B) Schematic depicting in trans mating scheme and phenotypic identification of germline SSA events. Percentage of F2 progeny exhibiting SSA-mediated transgene excision by DR length, confirmed through wild-type body color and the presence of the engineered silent mutation (TcG). Each dot represents a separate pair-mated cross (mean and ± s.e.m. are shown in red), with the sex of the F1 yReMET fly shown above. (C) Schematic depicting in cis mating scheme and phenotypic identification of germline SSA events. Percentage of F1 progeny exhibiting SSA-mediated transgene excision by DR length, confirmed through wild-type body color and the presence of the engineered silent mutation (TcG). Each dot represents a separate pair-mated cross (mean and ± s.e.m. are shown in red). UTR = Untranslated Region; DSB = Double Strand Break; UASp = Upstream Activation Sequence with pTransposase promoter; EGFP = Enhanced Green Fluorescent Protein; RFP = Red Fluorescent Protein; SSA = Single Strand Annealing; p values = * < 0.5, ** < 0.01, *** < 0.001, and **** < 0.0001; ns = not significant.

Fig 4. Repeat Mediated Excision of a Drive Element (ReMEDE).

(A) Schematic depicting ReMEDE construct and its removal by SSA. The construct contains components of both the ReMET (in cis) and yMCR constructs, including those essential for gene drive, i.e., Cas9 (under the control of a vasa promoter) and an sgRNA targeting exon 2 of the wild-type yellow gene (under the control of a U6:3 promoter). The core gene drive components are flanked by a pair of gypsy insulator sequences (gyp). Expression of I-SceI from nos-GS generates a DSB at the I-Site located between the DR, resulting in excision of the transgene by SSA, which is confirmed by the presence of an engineered silent mutation, TcG. (B) Schematic depicting maternal inheritance of an X-linked transgene or gene drive lacking ReMEDE. Mendelian inheritance of the recessive X-linked yellow mutant allele (generated through transgene insertion) from a heterozygous mother produces wild-type body pigmentation in heterozygous female progeny, but a yellow phenotype in the hemizygous male progeny. (C) When the transgene is also a gene drive, multiple Mendelian outcomes, as well as non-Mendelian exceptions, are possible. When the heterozygous drive allele present in the F1 female creates a DSB in its wild-type counterpart, HR-mediated repair results in allelic conversion increasing transgene frequency and generating super-Mendelian patterns of inheritance (green eyes). However, DSBs repaired by the more erroneous NHEJ may generate either in- or out-of-frame recessive null alleles, or dominant functional in-frame alleles. Non-Mendelian exceptions may also occur due to the zygotic perdurance of maternally deposited Cas9 RNPs generating DSBs in the paternal wild-type alleles, resulting in either in-frame or out-of-frame repair, which generates the additional possibilities illustrated. Theoretically, no wild-type X alleles should exist in the progeny of a cross involving a gene drive, which is why they are not shown. Maternal inheritance of the yMCR and yReMEDE transgenes. The percentage of progeny exhibiting drive, wild-type, or yellow phenotypes in replicate pair-mated crosses of heterozygous F1 females with wild-type males are shown. Each dot represents a separate pair-mated cross (D) Frequencies of out-of-frame indels in yMCR and yReMEDE progeny. Percentage of progeny by sex exhibiting a yellow body phenotype in pair-mated crosses involving either F1 yMCR or yReMEDE females and wild-type males. (E) Sequencing results for the yellow gene in wild-type progeny of pair-mated crosses involving F1 yMCR females and wild-type males, sorted by parental lineage. (F) Sequencing results for the yellow gene in wild-type progeny of pair-mated or en masse crosses involving F1 yReMEDE females and wild-type males, sorted by parental lineage. The sequence present at the PAM site is shown, as well as the DNA repair pathway presumed to be responsible for its generation. NHEJ = Non-Homologous End Joining; SSA = Single Strand Annealing; p values = ** < 0.01, *** < 0.001, and **** < 0.0001; ns = not significant (Wilcoxon signed-rank test).

Fig 5. I-Site stability and population cage trials with either yMCR or yReMEDE gene drives.

(A) Representative sequences over time at the I-SceI (I-Site) target site in the yReMEDE line (actual sequences are provided as a SAM file in supplementary information in S8 Fig). The samples column indicates the number of individuals sequenced on a particular date, with exception of the most recent samples, which represent 3 different mosquito pools, each consisting of 15 males and 15 females sequenced by nanopore. (B) Graphs show the prevalence of drive and wild type phenotypes (exhibited by both male and female flies) over time in cage trials involving either yMCR (green lines) or (C) yReMEDE (red lines). Solid lines represent single replicate cages, while dotted lines represent mean values.

Fig 6. Stochastic modeling of ReMEDE technology performance in a population.

All models were run with the X-linked inheritance module that outputs allele frequencies in two plots. One plot shows five simulations randomly selected from 100 independently run simulations (Left), while the other plot shows the mean of all simulations (Right). All simulations were run in a low threshold release scenario (10% of flies containing a drive allele) and in the absence of mating costs. (A) Simulation of yMCR gene drive, generating resistant alleles at frequencies comparable to those observed experimentally, but in the absence of reduced courtship activity and mating success. (B) Simulation of yReMEDE where SSA converts approximately 10% of the drive alleles to wild type. The generation of resistant alleles and other parameters occur at frequencies comparable to those shown in A. (C) Simulation of a gene drive containing ReMEDE that generates no resistant alleles, but with SSA converting approximately 30% of the drive alleles to wild type; a quarter of which retain the engineered resistant allele in the PAM site (ε = 0.25). (D) Simulation of a gene drive containing ReMEDE that generates no resistant alleles, but with SSA converting approximately 10% of the drive alleles to wild type, all of which retain the engineered resistant allele in the PAM site (ε = 1) (E) Simulation of a gene drive containing ReMEDE that generates no resistant alleles, but with SSA converting a modest ~5% of the drive alleles to wild type, a quarter of which retain the engineered resistant allele in the PAM site (ε = 0.25).