Publications by authors named "Anna Buchman"

17 Publications

  • Page 1 of 1

Engineered reproductively isolated species drive reversible population replacement.

Nat Commun 2021 06 2;12(1):3281. Epub 2021 Jun 2.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, San Diego, CA, USA.

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
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http://dx.doi.org/10.1038/s41467-021-23531-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173020PMC
June 2021

Inherently confinable split-drive systems in Drosophila.

Nat Commun 2021 03 5;12(1):1480. Epub 2021 Mar 5.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

CRISPR-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.
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http://dx.doi.org/10.1038/s41467-021-21771-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935863PMC
March 2021

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx.

J Vis Exp 2021 02 5(168). Epub 2021 Feb 5.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California;

CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.
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http://dx.doi.org/10.3791/62154DOI Listing
February 2021

Interdisciplinary development of a standardized introduction to gene drives for lay audiences.

BMC Med Res Methodol 2020 11 5;20(1):273. Epub 2020 Nov 5.

Department of Psychiatry, University of California, San Diego, 9500 Gilman Drive, MC 0811, La Jolla, California, 92093-0811, USA.

Background: While there is wide consensus that the public should be consulted about emerging technology early in development, it is difficult to elicit public opinion about innovations unfamiliar to lay audiences. We sought public input on a program of research on genetic engineering to control mosquito vectors of disease that is led by scientists at the University of California and funded by the U.S. Defense Advanced Research Projects Agency (DARPA). In preparation for this effort, we developed a series of narrated slideshows to prompt responses to the development of gene drive mosquito control strategies among lay people. We describe the development and content of these slideshows and evaluate their ability to elicit discussions among focus group participants.

Methods: In developing these materials, we used an iterative process involving input from experts in molecular genetics and vector control. Topics were chosen for their relevance to the goals of the scientists leading the program of research. Significant time was devoted to crafting explanations that would be accessible to uninitiated members of the public but still represent the science accurately. Through qualitative analysis of focus group discussions prompted by the slideshows, we evaluated the success of these slideshows in imparting clear technical information sufficient to inform lay discussion.

Results: The collaboration resulted in a series of four narrated slideshows that were used to anchor discussions in online focus groups. Many participants described the slideshows as interesting and informative, while also raising concerns and possible risks that were not directly addressed in the material presented. Open-ended comments from participants suggest that the slideshows inspired critical questions, reflection, and conversation about genetically engineered and gene drive mosquitoes. After the final and most technically complex slideshow, however, some respondents made comments suggestive of overwhelm or confusion.

Conclusion: Our narrated slideshows prompted engaged conversations about genetically engineered mosquitoes among members of the public who were generally naïve to this technology. Narrated slideshows may serve as viable and useful tools for future public engagement on other controversial emerging medical and public health technologies.
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http://dx.doi.org/10.1186/s12874-020-01146-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643426PMC
November 2020

Resistance to natural and synthetic gene drive systems.

J Evol Biol 2020 10 24;33(10):1345-1360. Epub 2020 Sep 24.

Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland.

Scientists are rapidly developing synthetic gene drive elements intended for release into natural populations. These are intended to control or eradicate disease vectors and pests, or to spread useful traits through wild populations for disease control or conservation purposes. However, a crucial problem for gene drives is the evolution of resistance against them, preventing their spread. Understanding the mechanisms by which populations might evolve resistance is essential for engineering effective gene drive systems. This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in general.
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http://dx.doi.org/10.1111/jeb.13693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796552PMC
October 2020

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

Mol Cell 2020 10 18;80(2):246-262.e4. Epub 2020 Sep 18.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA; Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA. Electronic address:

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
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http://dx.doi.org/10.1016/j.molcel.2020.09.003DOI Listing
October 2020

Programmable RNA Targeting Using CasRx in Flies.

CRISPR J 2020 06;3(3):164-176

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, USA.

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism . By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in , paving the way for future optimization of the system.
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http://dx.doi.org/10.1089/crispr.2020.0018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307691PMC
June 2020

Broad dengue neutralization in mosquitoes expressing an engineered antibody.

PLoS Pathog 2020 01 16;16(1):e1008103. Epub 2020 Jan 16.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.
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http://dx.doi.org/10.1371/journal.ppat.1008103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964813PMC
January 2020

Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.

G3 (Bethesda) 2020 02 6;10(2):827-837. Epub 2020 Feb 6.

Section of Cell and Developmental Biology and

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.
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http://dx.doi.org/10.1534/g3.119.400985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003086PMC
February 2020

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies.

BMC Neurosci 2019 06 17;20(1):27. Epub 2019 Jun 17.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 92093, USA.

Background: The mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as responding motor outputs, there has been a lack of studies able to monitor both concurrently.

Results: To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution.

Conclusions: By coupling in vivo live calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.
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http://dx.doi.org/10.1186/s12868-019-0511-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580577PMC
June 2019

Engineered resistance to Zika virus in transgenic expressing a polycistronic cluster of synthetic small RNAs.

Proc Natl Acad Sci U S A 2019 02 5;116(9):3656-3661. Epub 2019 Feb 5.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093;

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in , ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.
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http://dx.doi.org/10.1073/pnas.1810771116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397566PMC
February 2019

Synthetically engineered gene drive system in the worldwide crop pest .

Proc Natl Acad Sci U S A 2018 05 17;115(18):4725-4730. Epub 2018 Apr 17.

Department of Entomology, University of California, Riverside, CA 92521;

Synthetic gene drive systems possess enormous potential to replace, alter, or suppress wild populations of significant disease vectors and crop pests; however, their utility in diverse populations remains to be demonstrated. Here, we report the creation of a synthetic gene drive system in a major worldwide crop pest, We demonstrate that this drive system, based on an engineered maternal "toxin" coupled with a linked embryonic "antidote," is capable of biasing Mendelian inheritance rates with up to 100% efficiency. However, we find that drive resistance, resulting from naturally occurring genetic variation and associated fitness costs, can be selected for and hinder the spread of such a drive. Despite this, our results suggest that this gene drive could maintain itself at high frequencies in a wild population and spread to fixation if either its fitness costs or toxin resistance were reduced, providing a clear path forward for developing future such systems in this pest.
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http://dx.doi.org/10.1073/pnas.1713139115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939061PMC
May 2018

Engineered Reciprocal Chromosome Translocations Drive High Threshold, Reversible Population Replacement in Drosophila.

ACS Synth Biol 2018 05 13;7(5):1359-1370. Epub 2018 Apr 13.

Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , California 91125 , United States.

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.
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http://dx.doi.org/10.1021/acssynbio.7b00451DOI Listing
May 2018

Overcoming evolved resistance to population-suppressing homing-based gene drives.

Sci Rep 2017 06 19;7(1):3776. Epub 2017 Jun 19.

Department of Entomology, Center for Disease Vector Research, and Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA.

The recent development of a CRISPR-Cas9-based homing system for the suppression of Anopheles gambiae is encouraging; 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 theoretically explore 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 four multiplexed gRNAs, a mosquito species could potentially be suppressed on a continental scale. We also demonstrate successful proof-of-principle use of multiplexed ribozyme flanked gRNAs to induce mutations in vivo in Drosophila melanogaster - a strategy that could readily be adapted to engineer stable, homing-based drives in relevant organisms.
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http://dx.doi.org/10.1038/s41598-017-02744-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476637PMC
June 2017

Cheating evolution: engineering gene drives to manipulate the fate of wild populations.

Nat Rev Genet 2016 Mar 15;17(3):146-59. Epub 2016 Feb 15.

Department of Entomology, University of California, Riverside, Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, California 92521, USA.

Engineered gene drives - the process of stimulating the biased inheritance of specific genes - have the potential to enable the spread of desirable genes throughout wild populations or to suppress harmful species, and may be particularly useful for the control of vector-borne diseases such as malaria. Although several types of selfish genetic elements exist in nature, few have been successfully engineered in the laboratory thus far. With the discovery of RNA-guided CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) nucleases, which can be utilized to create, streamline and improve synthetic gene drives, this is rapidly changing. Here, we discuss the different types of engineered gene drives and their potential applications, as well as current policies regarding the safety and regulation of gene drives for the manipulation of wild populations.
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http://dx.doi.org/10.1038/nrg.2015.34DOI Listing
March 2016

Semele: a killer-male, rescue-female system for suppression and replacement of insect disease vector populations.

Genetics 2011 Feb 15;187(2):535-51. Epub 2010 Nov 15.

Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, London, W2 1PG, United Kingdom.

Two strategies to control mosquito-borne diseases, such as malaria and dengue fever, are reducing mosquito population sizes or replacing populations with disease-refractory varieties. We propose a genetic system, Semele, which may be used for both. Semele consists of two components: a toxin expressed in transgenic males that either kills or renders infertile wild-type female recipients and an antidote expressed in females that protects them from the effects of the toxin. An all-male release results in population suppression because wild-type females that mate with transgenic males produce no offspring. A release that includes transgenic females results in gene drive since females carrying the allele are favored at high population frequencies. We use simple population genetic models to explore the utility of the Semele system. We find that Semele can spread under a wide range of conditions, all of which require a high introduction frequency. This feature is desirable since transgenic insects released accidentally are unlikely to persist, transgenic insects released intentionally can be spatially confined, and the element can be removed from a population through sustained release of wild-type insects. We examine potential barriers to Semele gene drive and suggest molecular tools that could be used to build the Semele system.
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http://dx.doi.org/10.1534/genetics.110.124479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030495PMC
February 2011