Molecular safeguarding of CRISPR gene drive experiments

Champer, Jackson; Chung, Joanie; Lee, Yoo Lim; Liu, Chen; Yang, Eun Kyung; Wen, Zhaoxin; Clark, Andrew G.; Messer, Philipp W.

doi:10.7554/elife.41439

Cited by 109 publications

(138 citation statements)

References 33 publications

(57 reference statements)

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“…This indicates that any contribution of deposited nuclease to homing of these alleles in the germline is at best minimal. This is in contrast to reports in Drosophila where use of the nanos promoter can lead to 'shadow drive' in which perduring maternally deposited Cas9 can cause in homing in the germline even in the absence of genetically encoded Cas9 [29,30].…”

Section: New Gene Drive Constructs Confer Significantly Less Fecunditcontrasting

confidence: 84%

“…The propensity for maternal and paternal deposition of the nuclease under control of the promoters of the drive constructs is an important variable that we have shown can vary greatly. There is also the possibility that both the end-joining/HDR ratio and the magnitude of parental effect might additionally be locus dependent to some extent [12,29]. Indeed when the zpg promoter was used to control expression of a homing based gene drive at the doublesex locus we noticed a paternal effect contributing to reduced fecundity that was not observed here [8].…”

Section: Discussionmentioning

confidence: 89%

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Regulation of gene drive expression increases invasive potential and mitigates resistance

Hammond

Karlsson

Morianou

et al. 2018

Preprint

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CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance. Endonuclease-based homing gene drivesGene drives based on site-specific endonucleases were first proposed over 15 years ago [3] and recent advances in CRISPR technology have led to several demonstrations that this endonuclease, which is easy to reprogram to recognise a genomic site of choice, can be repurposed as a gene drive [4,5].The premise is that the endonuclease is sufficiently specific to recognise a DNA target sequence within a region of interest and the gene encoding the endonuclease is inserted within this target sequence on the chromosome, thereby rendering it immune to further cleavage. When a chromosome containing the endonuclease is paired with a chromosome containing the wild type target site, the site is cleaved to create a double stranded break (DSB) that can be repaired, either through simple 'cut and shut' nonhomologous end-joining (NHEJ) or through homology-directed repair (HDR). HDR involves strand invasion from the broken strand into regions of immediate homology on the intact chromosome, and synthesis across the intervening region to repair the gap. In the arrangement described this can lead to copying of the endonuclease, and its associated allele, from one chromosome to another in a process referred to as 'homing'. If homing takes place in the ge...

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Section: New Gene Drive Constructs Confer Significantly Less Fecunditcontrasting

confidence: 84%

Section: Discussionmentioning

confidence: 89%

Regulation of gene drive expression increases invasive potential and mitigates resistance

Hammond

Karlsson

Morianou

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…In fact, proof-of-concept CRISPR-based homing gene drives have recently been developed in several organisms including yeast (29)(30)(31)(32), flies (33)(34)(35)(36)(37)(38), mice (39), and two malaria vector species, Anopheles gambiae (40)(41)(42) and Anopheles stephensi (43).…”

Section: Introductionmentioning

confidence: 99%

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

Li¹,

Yang²,

Kandul³

et al. 2019

Preprint

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Aedes aegypti, the principal mosquito vector for many arboviruses that causes yellow fever, dengue, Zika, and chikungunya, increasingly infects millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has recently sparked significant enthusiasm for the genetic control of mosquito populations, however no such system has been developed in Ae. aegypti. To fill this void and demonstrate efficacy in Ae. aegypti, here we develop several CRISPR-based split-gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, 2 mathematical models predict that these systems could spread anti-pathogen effector genes into wild Ae. aegypti populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effectorlinked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission. Significance StatementAe. aegypti is a globally distributed arbovirus vector spreading deadly pathogens to millions of people annually. Current control methods are inadequate and therefore new technologies need to be innovated and implemented. With the aim of providing new tools for controlling this pest, here we engineered and tested several split gene drives in this species. These drives functioned at very high efficiency and may provide a tool to fill the void in controlling this vector. Taken together, our results provide compelling path forward for the feasibility of future effector-linked split-drive technologies that can contribute to the safe, sustained control and potentially the elimination of pathogens transmitted by this species.

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“…2The gRNA-only gene drive (gRNA GD) is based on CopyCat gRNA elements 19 that are capable of allelic conversion in the presence of a separate genetic source of Cas9. Since only the gRNA element is propagated in this case, its spread is regulated by the presence of a separate, static Cas9 transgene 10,[19][20][21] . The use of a full GD is causing concern to the scientific community as an accidental release could spread unchecked 15 .…”

mentioning

confidence: 99%

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment

et al. 2020

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CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.

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Molecular safeguarding of CRISPR gene drive experiments

Cited by 109 publications

References 33 publications

Regulation of gene drive expression increases invasive potential and mitigates resistance

Regulation of gene drive expression increases invasive potential and mitigates resistance

Development of a Confinable Gene-Drive System in the Human Disease Vector,Aedes aegypti

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment

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