scholarly journals Weakly deleterious natural genetic variation amplifies probability of resistance in multiplexed gene drive systems

2021 ◽  
Author(s):  
Bhavin S Khatri ◽  
Austin Burt
Keyword(s):  
De Novo ◽  
Large Population ◽  
Stochastic Simulations ◽  
Gene Drive ◽  
End Joining ◽  
Single Nucleotide ◽  
Major Barrier ◽  
Standing Variation ◽  
Large Populations ◽  
Drive Systems

Evolution of resistance is a major barrier to successful deployment of gene drive systems to suppress natural populations. Multiplexed guide RNAs that require resistance mutations in all target cut sites is a promising strategy to overcome resistance. Using novel stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) non-homologous end-joining mutations, 2) single nucleotide mutants arising de novo or, 3) single nucleotide polymorphisms pre-existing as standing variation. If the fraction of functional end-joining mutants is rare, we show that standing variation dominates, via a qualitatively new phenomenon where weakly deleterious variants significantly amplify the probability of multi-site resistance. This means resistance can be probable even with many target sites in not very large populations. This result has broad application to resistance arising in multi-site evolutionary scenarios including the evolution of vaccine escape mutations in large populations.

scholarly journals Current CRISPR gene drive systems are likely to be highly invasive in wild populations

2017 ◽  
Author(s):  
Charleston Noble ◽  
Ben Adlam ◽  
George M. Church ◽  
Kevin M. Esvelt ◽  
Martin A. Nowak
Keyword(s):  
Local Population ◽  
Gene Drive ◽  
Host Organism ◽  
Wild Populations ◽  
Flow Rates ◽  
Standing Variation ◽  
Large Populations ◽  
Mitigating Factors ◽  
Drive Systems ◽  
Gene Drives

AbstractRecent reports have suggested that CRISPR-based gene drives are unlikely to invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption. We show that although resistance prevents drive systems from spreading to fixation in large populations, even the least effective systems reported to date are highly invasive. Releasing a small number of organisms often causes invasion of the local population, followed by invasion of additional populations connected by very low gene flow rates. Examining the effects of mitigating factors including standing variation, inbreeding, and family size revealed that none of these prevent invasion in realistic scenarios. Highly effective drive systems are predicted to be even more invasive. Contrary to the National Academies report on gene drive, our results suggest that standard drive systems should not be developed nor field-tested in regions harboring the host organism.

scholarly journals Fast neutron mutagenesis in soybean enriches for small indels and creates frameshift mutations

2021 ◽  
Author(s):  
Skylar R Wyant ◽  
M Fernanda Rodriguez ◽  
Corey K Carter ◽  
Wayne A Parrott ◽  
Scott A Jackson ◽  
...  
Keyword(s):  
Fast Neutron ◽  
De Novo ◽  
Spontaneous Mutation ◽  
Local Context ◽  
Induced Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Frameshift Mutations ◽  
Small Indels ◽  
Standing Variation

Abstract The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single nucleotide variants. The accumulation of new single nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1–6 base pairs, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss of function mutations that impact single genes.

scholarly journals Local ancestry analysis reveals genomic convergence in extremophile fishes

2019 ◽  
Vol 374 (1777) ◽  
pp. 20180240 ◽  
Author(s):  
Anthony P. Brown ◽  
Kerry L. McGowan ◽  
Enrique J. Schwarzkopf ◽  
Ryan Greenway ◽  
Lenin Arias Rodriguez ◽  
...  
Keyword(s):  
Candidate Genes ◽  
De Novo ◽  
Large Population ◽  
Significant Proportion ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations ◽  
Genetic Constraints ◽  
Standing Variation ◽  
Whole Genome Resequencing ◽  
Genomic Convergence

The molecular basis of convergent phenotypes is often unknown. However, convergence at a genomic level is predicted when there are large population sizes, gene flow among diverging lineages or strong genetic constraints. We used whole-genome resequencing to investigate genomic convergence in fishes ( Poecilia spp.) that have repeatedly colonized hydrogen sulfide (H 2 S)-rich environments in Mexico. We identified genomic similarities in both single nucleotide polymorphisms (SNPs) and structural variants (SVs) among independently derived sulfide spring populations, with approximately 1.2% of the genome being shared among sulfidic ecotypes. We compared these convergent genomic regions to candidate genes for H 2 S adaptation identified from transcriptomic analyses and found that a significant proportion of these candidate genes (8%) were also in regions where sulfidic individuals had similar SNPs, while only 1.7% were in regions where sulfidic individuals had similar SVs. Those candidate genes included genes involved in sulfide detoxification, the electron transport chain (the main toxicity target of H 2 S) and other processes putatively important for adaptation to sulfidic environments. Regional genomic similarity across independent populations exposed to the same source of selection is consistent with selection on standing variation or introgression of adaptive alleles across divergent lineages. However, combined with previous analyses, our data also support that adaptive changes in mitochondrially encoded subunits arose independently via selection on de novo mutations. Pressing questions remain on what conditions ultimately facilitate the independent rise of adaptive alleles at the same loci in separate populations, and thus, the degree to which evolution is repeatable or predictable. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions'.

scholarly journals Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 172-179 ◽  
Author(s):  
Rebeca Carballar-Lejarazú ◽  
Adam Kelsey ◽  
Thai Binh Pham ◽  
Eric P Bennett ◽  
Anthony A James
Keyword(s):  
Anopheles Stephensi ◽  
Field Testing ◽  
Nonhomologous End Joining ◽  
Mosquito Vector ◽  
Gene Drive ◽  
End Joining ◽  
Indel Detection ◽  
Vector Mosquito ◽  
Droplet Pcr ◽  
Drive Systems

CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.

scholarly journals Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

2015 ◽  
Vol 112 (13) ◽  
pp. 4038-4043 ◽  
Author(s):  
Sanjay Basu ◽  
Azadeh Aryan ◽  
Justin M. Overcash ◽  
Glady Hazitha Samuel ◽  
Michelle A. E. Anderson ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Specific Gene ◽  
Gene Drive ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Essential Components ◽  
Drive Systems

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

Natural Selection in Large Populations

2020 ◽  
pp. 109-146
Author(s):  
Daniel L. Hartl
Keyword(s):  
Natural Selection ◽  
Balancing Selection ◽  
Large Population ◽  
Natural Populations ◽  
Meiotic Drive ◽  
Gene Drive ◽  
Background Selection ◽  
Large Populations ◽  
Gametic Selection ◽  
Soft Selective Sweeps

This chapter includes selection in haploid and diploid organisms, hard and soft selective sweeps, background selection, and the probability of ultimate survival of a new favorable mutation in a large population. It considers overdominance and heterozygote inferiority in detail as well as different types of equilibria and the fundamental theorem of natural selection. Various types of balancing selection are examined including mutation–selection balance, migration–selection balance, meiotic drive and gametic selection, and the theory of CRISPR-mediated gene drive to control natural populations. It closes with a discussion of other modes of selection and their implications.

scholarly journals Inherently confinable split-drive systems in Drosophila

2020 ◽  
Author(s):  
Gerard Terradas ◽  
Anna B. Buchman ◽  
Jared B. Bennett ◽  
Isaiah Shriner ◽  
John M. Marshall ◽  
...  
Keyword(s):  
Gene Drive ◽  
End Joining ◽  
Single Generation ◽  
Nhej Pathway ◽  
Endogenous Gene ◽  
Homology Directed Repair ◽  
Split Gene ◽  
Target Chromosome ◽  
Non Homologous End Joining ◽  
Drive Systems

AbstractCRISPR-based gene drive systems, which copy themselves based on gene conversion mediated by the homology directed repair (HDR) pathway, have potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway that are rendered resistant to Cas9 cleavage can interrupt the spread of genedrive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences to restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles, combined with recessive Mendelian processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that were inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multi-generational cage trials, sGD follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage or lethal/sterile mosaic phenotypes, leading to inherently confineable drive outcomes.

scholarly journals Inherently confinable split-drive systems in Drosophila

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gerard Terradas ◽  
Anna B. Buchman ◽  
Jared B. Bennett ◽  
Isaiah Shriner ◽  
John M. Marshall ◽  
...  
Keyword(s):  
Gene Drive ◽  
End Joining ◽  
Single Generation ◽  
Nhej Pathway ◽  
Endogenous Gene ◽  
Homology Directed Repair ◽  
Split Gene ◽  
Target Chromosome ◽  
Non Homologous End Joining ◽  
Drive Systems

AbstractCRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

scholarly journals Current CRISPR gene drive systems are likely to be highly invasive in wild populations

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Charleston Noble ◽  
Ben Adlam ◽  
George M Church ◽  
Kevin M Esvelt ◽  
Martin A Nowak
Keyword(s):  
Gene Flow ◽  
Empirical Data ◽  
Local Population ◽  
Malaria Prevention ◽  
Field Trials ◽  
Gene Drive ◽  
Wild Populations ◽  
Target Species ◽  
Large Populations ◽  
Drive Systems

Recent reports have suggested that self-propagating CRISPR-based gene drive systems are unlikely to efficiently invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption for population alteration drives. Our models show that although resistance prevents spread to fixation in large populations, even the least effective drive systems reported to date are likely to be highly invasive. Releasing a small number of organisms will often cause invasion of the local population, followed by invasion of additional populations connected by very low rates of gene flow. Hence, initiating contained field trials as tentatively endorsed by the National Academies report on gene drive could potentially result in unintended spread to additional populations. Our mathematical results suggest that self-propagating gene drive is best suited to applications such as malaria prevention that seek to affect all wild populations of the target species.

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