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.

scholarly journals A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population

2020 ◽  
Vol 117 (39) ◽  
pp. 24377-24383 ◽  
Author(s):  
Jackson Champer ◽  
Emily Yang ◽  
Esther Lee ◽  
Jingxian Liu ◽  
Andrew G. Clark ◽  
...  
Keyword(s):  
Gene Drive ◽  
End Joining ◽  
Large Cage ◽  
Vector Borne Diseases ◽  
Guide Rnas ◽  
Genetic Modifications ◽  
New Strategy ◽  
Cage Population ◽  
Vector Borne ◽  
Gene Drives

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Gene Function ◽  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
Complex Activity ◽  
Guide Rnas ◽  
Carry Over ◽  
Population Suppression

AbstractA gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single gRNA that together target a specific gene for cleavage. Homing was observed, but resistant alleles, immune to cleavage, while retaining wildtype gene function, were also created through non-homologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs, targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and guide RNAs (gRNAs) designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments the HEGs failed to spread, due to the high fitness load induced in offspring as a result of maternal carry over of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.Significance statementHEG-based gene drive can bring about population suppression when genes required for viability or fertility are targeted. However, these strategies are vulnerable to failure through mechanisms that create alleles resistant to cleavage, but that retain wildtype gene function. We show that resistance allele creation can be prevented through the use of gRNAs designed to cleave a gene at four target sites. However, homing rates were modest, and the HEGs were unstable during homing. In addition, use of a promoter active in the female germline resulted in levels of HEG carryover that compromised the viability or fertility of HEG-bearing heterozygotes, thereby preventing drive. We propose strategies that can help to overcome these problems in next generation HEG systems.

scholarly journals Modeling confinement and reversibility of threshold-dependent gene drive systems in spatially-explicit Aedes aegypti populations

2019 ◽  
Author(s):  
Héctor M. Sánchez C. ◽  
Jared B. Bennett ◽  
Sean L. Wu ◽  
Gordana Rašić ◽  
Omar S. Akbari ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Disease Transmission ◽  
Release Site ◽  
Field Trials ◽  
Mosquito Vector ◽  
Gene Drive ◽  
Isolated Populations ◽  
Dependent Behavior ◽  
Wide Scale ◽  
Drive Systems

AbstractBackgroundThe discovery of CRISPR-based gene editing and its application to homing-based gene drive systems has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently-engineered threshold-dependent gene drive systems - reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UDMEL - to explore their ability to be confined and remediated.ResultsWe simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UDMEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely.ConclusionsOur analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.

scholarly journals Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

2016 ◽  
Vol 82 (16) ◽  
pp. 4876-4895 ◽  
Author(s):  
Adam W. Westbrook ◽  
Murray Moo-Young ◽  
C. Perry Chou
Keyword(s):  
Bacillus Subtilis ◽  
Gene Mutations ◽  
Biochemical Properties ◽  
Site Specific ◽  
Content Type ◽  
Host System ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Transcriptional Modulation ◽  
Short Palindromic Repeat

ABSTRACTThe establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction inBacillus subtilisis essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering inB. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed forEscherichia coliandSaccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery.IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system inBacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering ofB. subtilisstrains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

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 Genomic insertion locus and Cas9 expression in the germline affect CRISPR/Cas9-based gene drive performance in the yellow fever mosquito Aedes aegypti

2021 ◽  
Author(s):  
William R Reid ◽  
Jingyi Lin ◽  
Adeline E Williams ◽  
Rucsanda Juncu ◽  
Ken E Olson ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Yellow Fever ◽  
Drive System ◽  
Gene Drive ◽  
Yellow Fever Mosquito ◽  
Genomic Locus ◽  
Population Replacement ◽  
Novel Approach ◽  
Drive Systems ◽  
Insertion Locus

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Here we report an autonomous single-component gene drive system in Ae. aegypti that is designed for persistent population replacement. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus be amenable to both gene drive and the appropriate expression of the antiviral effector. In our study, we took a reverse engineering approach to target two genomic loci ideal for the expression of antiviral effectors and further investigated the use of three promoters for Cas9 expression (nanos, β2-tubulin, or zpg) for the gene drive. We found that both promoter selection and genomic target site strongly influenced the efficiency of the drive, resulting in 100% inheritance in some crosses. We also observed the formation of inheritable gene drive blocking indels (GDBI) in the genomic locus with the highest levels of gene drive. Overall, our drive system forms a platform for the further testing of driving antipathogen effector genes through Ae. aegypti populations.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Vol 115 (40) ◽  
pp. E9343-E9352 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
End Joining ◽  
Complex Activity ◽  
Guide Rna ◽  
Type Gene ◽  
Population Suppression

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs inDrosophila melanogastertargeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.

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