scholarly journals Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

PLoS Genetics ◽  
2021 ◽  
Vol 17 (10) ◽  
pp. e1009740
Author(s):  
Silke Fuchs ◽  
William T. Garrood ◽  
Anna Beber ◽  
Andrew Hammond ◽  
Roberto Galizi ◽  
...  
Keyword(s):  
Essential Gene ◽  
Target Site ◽  
Target Sequence ◽  
Lethal Gene ◽  
Gene Drive ◽  
Continuous Sampling ◽  
Conserved Sequence ◽  
A Site ◽  
Gene Drives

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

scholarly journals Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

2021 ◽  
Author(s):  
Silke Fuchs ◽  
William T. Garrood ◽  
Anna Beber ◽  
Andrew Hammond ◽  
Roberto Galizi ◽  
...  
Keyword(s):  
Essential Gene ◽  
Target Site ◽  
Target Sequence ◽  
Gene Drive ◽  
Continuous Sampling ◽  
Conserved Sequence ◽  
Target Site Resistance ◽  
A Site ◽  
Gene Drives

CRISPR-based homing gene drives designed to disrupt essential genes whilst biasing their own inheritance can suppress mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is usually a reliable indicator that there is functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector An. gambiae targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development and which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

scholarly journals Regulation of gene drive expression increases invasive potential and mitigates resistance

2018 ◽  
Author(s):  
Andrew Hammond ◽  
Xenia Karlsson ◽  
Ioanna Morianou ◽  
Kyros Kyrou ◽  
Andrea Beaghton ◽  
...  
Keyword(s):  
First Generation ◽  
Target Site ◽  
Regulatory Sequences ◽  
Target Sequence ◽  
Mosquito Vector ◽  
Gene Drive ◽  
Dna Breaks ◽  
Target Site Resistance ◽  
The Impact ◽  
Gene Drives

AbstractCRISPR-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.

scholarly journals Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

1986 ◽  
Vol 6 (7) ◽  
pp. 2482-2489 ◽  
Author(s):  
B J Andrews ◽  
M McLeod ◽  
J Broach ◽  
P D Sadowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Core Region ◽  
Target Sequence ◽  
Repeat Elements ◽  
Flp Recombinase ◽  
Target Sequences ◽  
2 Micron Plasmid ◽  
A Site

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.

scholarly journals Integral Gene Drives: an “operating system” for population replacement

2018 ◽  
Author(s):  
Alexander Nash ◽  
Giulia Mignini Urdaneta ◽  
Andrea K. Beaghton ◽  
Astrid Hoermann ◽  
Philippos Aris Papathanos ◽  
...  
Keyword(s):  
Field Testing ◽  
Target Site ◽  
Gene Drive ◽  
Population Replacement ◽  
Pathogen Effector ◽  
Site Variation ◽  
The Face ◽  
Endogenous Genes ◽  
Target Site Resistance ◽  
Gene Drives

AbstractFirst generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.

scholarly journals Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

2018 ◽  
Author(s):  
Megan E. Goeckel ◽  
Erianna M. Basgall ◽  
Isabel C. Lewis ◽  
Samantha C. Goetting ◽  
Yao Yan ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Gene Drive ◽  
Wild Populations ◽  
Vector Borne Diseases ◽  
Vector Borne ◽  
Mendelian Laws ◽  
Artificial Gene ◽  
Nuclear Localization Sequences ◽  
Gene Drives

ABSTRACTThe bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. In this study, we use an artificial gene drive system in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences on Cas9 fusion proteins in vivo and demonstrate that appended signals can titrate gene drive activity and serve as a potential molecular safeguard.

scholarly journals Gene-drive-mediated extinction is thwarted by population structure and evolution of sib mating

2019 ◽  
Vol 2019 (1) ◽  
pp. 66-81 ◽  
Author(s):  
James J Bull ◽  
Christopher H Remien ◽  
Stephen M Krone
Keyword(s):  
Population Structure ◽  
Genome Engineering ◽  
Random Mating ◽  
Structured Populations ◽  
Target Sequence ◽  
Gene Drive ◽  
Resistance Evolution ◽  
Sib Mating ◽  
Mean Fitness ◽  
Gene Drives

AbstractBackground and objectivesGenetic engineering combined with CRISPR technology has developed to the point that gene drives can, in theory, be engineered to cause extinction in countless species. Success of extinction programs now rests on the possibility of resistance evolution, which is largely unknown. Depending on the gene-drive technology, resistance may take many forms, from mutations in the nuclease target sequence (e.g. for CRISPR) to specific types of non-random population structures that limit the drive (that may block potentially any gene-drive technology).MethodologyWe develop mathematical models of various deviations from random mating to consider escapes from extinction-causing gene drives. A main emphasis here is sib mating in the face of recessive-lethal and Y-chromosome drives.ResultsSib mating easily evolves in response to both kinds of gene drives and maintains mean fitness above 0, with equilibrium fitness depending on the level of inbreeding depression. Environmental determination of sib mating (as might stem from population density crashes) can also maintain mean fitness above 0. A version of Maynard Smith’s haystack model shows that pre-existing population structure can enable drive-free subpopulations to be maintained against gene drives.Conclusions and implicationsTranslation of mean fitness into population size depends on ecological details, so understanding mean fitness evolution and dynamics is merely the first step in predicting extinction. Nonetheless, these results point to possible escapes from gene-drive-mediated extinctions that lie beyond the control of genome engineering.Lay summaryRecent gene drive technologies promise to suppress and even eradicate pests and disease vectors. Simple models of gene-drive evolution in structured populations show that extinction-causing gene drives can be thwarted both through the evolution of sib mating as well as from purely demographic processes that cluster drive-free individuals.

scholarly journals Molecular safeguarding of CRISPR gene drive experiments

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jackson Champer ◽  
Joan Chung ◽  
Yoo Lim Lee ◽  
Chen Liu ◽  
Emily Yang ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Natural Population ◽  
Early Embryo ◽  
Drive System ◽  
Target Site ◽  
Gene Drive ◽  
Gene Drives ◽  
Synthetic Target

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.

scholarly journals Overcoming evolved resistance to population-suppressing homing-based gene drives

2016 ◽  
Author(s):  
John M. Marshall ◽  
Anna Buchman ◽  
Héctor M. Sánchez C. ◽  
Omar S. Akbari
Keyword(s):  
Mosquito Species ◽  
Gene Drive ◽  
Resistant Allele ◽  
Guide Rnas ◽  
Continental Scale ◽  
Fitness Advantage ◽  
Health Related ◽  
Drive Systems ◽  
Gene Drives

AbstractThe use of homing-based gene drive systems to modify or suppress wild populations of a given species has been proposed as a solution to a number of significant ecological and public health related problems, including the control of mosquito-borne diseases. The recent development of a CRISPR-Cas9-based homing system for the suppression ofAnopheles gambiae, the main African malaria vector, is encouraging for this approach; however, with current designs, the slow emergence of homing-resistant alleles is expected to result in suppressed populations rapidly rebounding, as homing-resistant alleles have a significant fitness advantage over functional, population-suppressing homing alleles. To explore this concern, we develop a mathematical model to estimate tolerable rates of homing-resistant allele generation to suppress a wild population of a given size. Our results suggest that, to achieve meaningful population suppression, tolerable rates of resistance allele generation are orders of magnitude smaller than those observed for current designs for CRISPR-Cas9-based homing systems. To remedy this, we propose a homing system architecture in which guide RNAs (gRNAs) are multiplexed, increasing the effective homing rate and decreasing the effective resistant allele generation rate. Modeling results suggest that the size of the population that can be suppressed increases exponentially with the number of multiplexed gRNAs and that, with six multiplexed gRNAs, a mosquito species could potentially be suppressed on a continental scale. We also demonstrate successful multiplexingin vivoinDrosophila melanogasterusing a ribozyme-gRNA-ribozyme (RGR) approach – a strategy that could readily be adapted to engineer stable, homing-based suppression drives in relevant organisms.

scholarly journals Gene-drive-mediated extinction is thwarted by evolution of sib mating

2019 ◽  
Author(s):  
James J Bull ◽  
Christopher H Remien ◽  
Stephen M Krone
Keyword(s):  
Genome Engineering ◽  
Random Mating ◽  
Target Sequence ◽  
Gene Drive ◽  
Resistance Evolution ◽  
Sib Mating ◽  
Population Structures ◽  
The Face ◽  
Mean Fitness ◽  
Gene Drives

AbstractGenetic engineering combined with CRISPR technology has developed to the point that gene drives can, in theory, be engineered to cause extinction in countless species. Success of extinction programs now rests on the possibility of resistance evolution, which is largely unknown. For CRISPR technology, resistance may take many forms, from mutations in the nuclease target sequence to specific types of non-random population structures that limit the drive. We develop mathematical models of various deviations from random mating to consider escapes from extinction-causing gene drives. We use a version of Maynard Smith’s haystack model to show that population structure can enable drive-free subpopulations to be maintained against gene drives. Our main emphasis, however, is sib mating in the face of recessive-lethal and Y-chromosome drives. Sib mating easily evolves in response to both kinds of gene drives and maintains mean fitness above 0, with equilibrium fitness depending on the level of inbreeding depression. Environmental determination of sib mating (as might stem from population density crashes) can also maintain mean fitness above 0. Translation of mean fitness into population size depends on ecological details, so understanding mean fitness evolution and dynamics is merely the first step in predicting extinction. Nonetheless, these results point to possible escapes from gene drive-mediated extinctions that lie beyond the control of genome engineering.

scholarly journals Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment

1996 ◽  
Vol 314 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Philip M. BROWN ◽  
Amelia DRABBLE ◽  
Keith R. FOX
Keyword(s):  
Target Site ◽  
Target Sequence ◽  
Similar Concentration ◽  
Helix Formation ◽  
Dnase I Footprinting ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Fragment ◽  
A Site ◽  
Formation Of Complexes

We have used DNase I footprinting to examine the effect of a triplex-binding ligand on the formation of parallel intermolecular DNA triple helices at a mixed sequence target site contained within a natural DNA fragment (tyrT). In the presence of 10 μM ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine), the binding of CTCTTTTTGCTT (12G) to the sequence GAGAAAAATGAA (generating a complex containing 8×T·AT, 1×G·TA and 3×C+·GC triplets) was enhanced 3-fold at pH 5.5. When the oligonucleotide CTCTTTTTTCTT (12T) was substituted for 12G (replacing G·TA with T·TA) there was a large reduction in affinity for the target sequence. However, this was stabilized by about 300-fold in the presence of the ligand, requiring a similar concentration to produce a footprint as 12G in the absence of the ligand. When the sequence of the target site was altered to GAGAAAAAAGAA, generating an uninterrupted run of purines [tyrT(46A)], the binding of 12T (generating a complex containing 9×T·AT, and 3×C+·GC triplets) was enhanced 3-fold by 10 μM of the triplex-binding ligand. However, although the binding of 12G to this sequence, generating a complex containing a G·AT triplet, was much weaker, this too was stabilized by about 30-fold by the ligand, requiring a similar concentration as the perfect matched oligonucleotide (12T) in the absence of the ligand. A secondary, less stable footprint was also observed in these fragments when using either 12T or 12G, which was evident only in the presence of the triplex-binding ligand. This site, which contained a number of triplet mismatches, appears to be related to the formation of four or five central T·AT triplets. This reduction in the stringency of oligonucleotide binding by the triplex-binding ligand promotes the formation of complexes at non-targeted regions but may also have the potential for enabling recognition at sites that contain regions where there are no specific triplet matches.

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