Gene drive and resilience through renewal with next generationCleave and Rescueselfish genetic elements

Abstract

Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. Here, we show that resilience can be achieved through cycles of population modification with “Cleave and Rescue” (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvRn+1, carries a Rescue transgene from an earlier element, ClvRn. ClvRn+1 should spread within a population of ClvRn, while also bringing about a decrease in its frequency. To test this hypothesis, we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.

Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive

“…A ClvR element [ 5 , 6 ]) (also known as Toxin Antidote Recessive Embryo (TARE) in a related proof-of-principle implementation [ 18 ]), which serves as the starting point for this work, is a self-sustaining gene drive element ( Fig 1A ). It consists of a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene (located anywhere in the genome) in the germline and in the zygote using Cas9/gRNAs carried over from the mother, and a tightly linked version of the essential gene recoded to be resistant to cleavage and ectopic gene conversion with the endogenous locus (the Rescue ) [ 5 , 6 , 18 ]. ClvR/TARE (hereafter referred to as ClvR ) spreads because Cas9/gRNAs create loss-of-function (LOF) alleles (the drive force) that select against those who fail to inherit ClvR in LOF homozygotes.…”

“…It lacks a release threshold when fitness costs are absent, but acquires one in their presence. When drive occurs, transgenes spread to genotype or allele fixation depending on the location of ClvR and the gene being targeted [ 5 , 6 , 18 , 20 ]. Finally, haploinsufficient or haplolethal genes can also be targeted.…”

“…Finally, haploinsufficient or haplolethal genes can also be targeted. Higher thresholds for drive are created, but when drive occurs, it still leads to transgene and/or allele fixation [ 5 , 6 , 18 , 20 ].…”

“…Split 50cM ClvR V2 (implemented in the experimental section below) is particularly easy to synthesize since organisms carrying each transgene-bearing component are homozygous viable in isolation. In contrast, for split 50cM ClvR V1 the Cas9/gRNA transgenes must be introduced into, and maintained within the Rescue /Cargo background, as with ClvR [ 5 , 6 ]. Versions of split ClvR in which there is linkage between the components ( Fig 1E , genetic map distance <50cM on the same chromosome) are discussed in a later section.…”

“…These are are strong drivers at low frequency and in the presence of significant fitness costs [ 14 , 15 ]. Medea [ 1 , 16 , 17 ] Cleave and Rescue ( ClvR ) [ 5 , 6 ], TARE [ 18 ] and a number of other proposed gene drive elements [ 19 , 20 ] are also low threshold. However, these are weak drivers at low frequency and acquire a threshold in the presence of any fitness costs.…”