Publications by authors named "Omar S Akbari"

71 Publications

Combating mosquito-borne diseases using genetic control technologies.

Nat Commun 2021 07 19;12(1):4388. Epub 2021 Jul 19.

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

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.
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http://dx.doi.org/10.1038/s41467-021-24654-zDOI Listing
July 2021

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

Suppression of female fertility in with a CRISPR-targeted male-sterile mutation.

Proc Natl Acad Sci U S A 2021 Jun;118(22)

Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106;

spread devastating viruses such as dengue, which causes disease among 100 to 400 million people annually. A potential approach to control mosquito disease vectors is the sterile insect technique (SIT). The strategy involves repeated release of large numbers of sterile males, which reduces insect populations because the sterile males mate and thereby suppress the fertility of females that would otherwise mate with fertile males. While SIT has been successful in suppressing certain agricultural pests, it has been less effective in depressing populations of This limitation is in part because of the fitness effects resulting from mutagenizing the mosquitoes nonspecifically. Here, we introduced and characterized the impact on female fertility of an mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the () gene, which eliminates male fertility. When we allowed wild-type females to first mate with mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced mutant and wild-type males simultaneously with wild-type females and found that a larger number of mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing sterile males to improve the efficacy of SIT in suppressing populations of through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.
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http://dx.doi.org/10.1073/pnas.2105075118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179229PMC
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

A confinable home-and-rescue gene drive for population modification.

Elife 2021 Mar 5;10. Epub 2021 Mar 5.

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

Homing-based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification home-and-rescue (HomeR) drive in targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.
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http://dx.doi.org/10.7554/eLife.65939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968924PMC
March 2021

Mechanistically comparing reproductive manipulations caused by selfish chromosomes and bacterial symbionts.

Heredity (Edinb) 2021 May 1;126(5):707-716. Epub 2021 Mar 1.

W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.

Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.
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http://dx.doi.org/10.1038/s41437-021-00410-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102561PMC
May 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

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

Genome Res 2021 Mar 8;31(3):512-528. Epub 2021 Jan 8.

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.
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http://dx.doi.org/10.1101/gr.265157.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919454PMC
March 2021

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis.

J Vis Exp 2020 12 4(166). Epub 2020 Dec 4.

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

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.
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http://dx.doi.org/10.3791/61892DOI Listing
December 2020

Targeting female flight for genetic control of mosquitoes.

PLoS Negl Trop Dis 2020 12 3;14(12):e0008876. Epub 2020 Dec 3.

Arthropod Genetics, The Pirbright Institute, Pirbright, United Kingdom.

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.
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http://dx.doi.org/10.1371/journal.pntd.0008876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714197PMC
December 2020

Opinion: Standardizing the definition of gene drive.

Proc Natl Acad Sci U S A 2020 12 18;117(49):30864-30867. Epub 2020 Nov 18.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093

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http://dx.doi.org/10.1073/pnas.2020417117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733814PMC
December 2020

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

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2.

medRxiv 2020 Oct 20. Epub 2020 Oct 20.

Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.
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http://dx.doi.org/10.1101/2020.10.14.20212795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587836PMC
October 2020

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways.

Cells 2020 09 27;9(10). Epub 2020 Sep 27.

Department of Biochemistry and Genome Science Institute, Boston University School of Medicine, Boston, MA 02118, USA.

Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.
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http://dx.doi.org/10.3390/cells9102180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601171PMC
September 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

Improved reference genome of the arboviral vector Aedes albopictus.

Genome Biol 2020 08 26;21(1):215. Epub 2020 Aug 26.

Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, 20892-2152, MD, USA.

Background: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes.

Results: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes.

Conclusion: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.
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http://dx.doi.org/10.1186/s13059-020-02141-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448346PMC
August 2020

A day in the life of a mosquito insectary team: pushing for solutions to mosquito-borne diseases.

Lab Anim (NY) 2020 09;49(9):241-243

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

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http://dx.doi.org/10.1038/s41684-020-0617-yDOI Listing
September 2020

Translating gene drive science to promote linguistic diversity in community and stakeholder engagement.

Glob Public Health 2020 10 26;15(10):1551-1565. Epub 2020 Jun 26.

The Qualcomm Institute, Calit2, UC San Diego, La Jolla, CA, USA.

Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.
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http://dx.doi.org/10.1080/17441692.2020.1779328DOI 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

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus.

J Vis Exp 2020 05 24(159). Epub 2020 May 24.

Section of Cell and Developmental Biology, University of California, San Diego; Tata Institute for Genetics and Society, University of California, San Diego;

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.
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http://dx.doi.org/10.3791/61375DOI Listing
May 2020

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

BMC Biol 2020 05 12;18(1):50. Epub 2020 May 12.

Division of Epidemiology and Biostatistics, School of Public Health, University of California, Berkeley, CA, 94720, USA.

Background: The 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 UD-to explore their ability to be confined and remediated.

Results: We 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. UD 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.

Conclusions: Our 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.
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http://dx.doi.org/10.1186/s12915-020-0759-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218562PMC
May 2020

A drug-inducible sex-separation technique for insects.

Nat Commun 2020 04 30;11(1):2106. Epub 2020 Apr 30.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 92092, USA.

Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.
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http://dx.doi.org/10.1038/s41467-020-16020-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193620PMC
April 2020

Genome elimination mediated by gene expression from a selfish chromosome.

Sci Adv 2020 04 3;6(14):eaaz9808. Epub 2020 Apr 3.

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

Numerous plants and animals harbor selfish B chromosomes that "drive" or transmit themselves at super-Mendelian frequencies, despite long-term fitness costs to the organism. Currently, it is unknown how B chromosome drive is mediated, and whether B-gene expression plays a role. We used modern sequencing technologies to analyze the fine-scale sequence composition and expression of paternal sex ratio (PSR), a B chromosome in the jewel wasp . PSR causes female-to-male conversion by destroying the sperm's hereditary material in young embryos to drive. Using RNA interference, we demonstrate that testis-specific expression of a PSR-linked gene, named , facilitates this genome elimination-and-sex conversion effect. encodes a putative protein with a DNA binding domain, suggesting a functional link with the sperm-derived chromatin.
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http://dx.doi.org/10.1126/sciadv.aaz9808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124933PMC
April 2020

Progress towards engineering gene drives for population control.

J Exp Biol 2020 02 7;223(Pt Suppl 1). Epub 2020 Feb 7.

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

Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.
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http://dx.doi.org/10.1242/jeb.208181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790193PMC
February 2020

The Developmental Transcriptome of , a Major Worldwide Human Disease Vector.

G3 (Bethesda) 2020 03 5;10(3):1051-1062. Epub 2020 Mar 5.

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

mosquitoes are important vectors for a number of human pathogens including the Zika, dengue, and chikungunya viruses. Capable of displacing populations, this mosquito adapts to cooler environments which increases its geographical range and transmission potential. There are limited control strategies for mosquitoes which is likely attributed to the lack of comprehensive biological studies on this emerging vector. To fill this void, here using RNAseq we characterized mRNA expression profiles at 34 distinct time points throughout development providing the first high-resolution comprehensive view of the developmental transcriptome of this worldwide human disease vector. This enabled us to identify several patterns of shared gene expression among tissues as well as sex-specific expression patterns. To illuminate the similarities and differences with , a related human disease vector, we also performed a comparative analysis between the two developmental transcriptomes, identifying life stages where the two species exhibit similar and distinct gene expression patterns. These findings provide insights into the similarities and differences between and mosquito biology. In summary, the results generated from this study should form the basis for future investigations on the biology of and provide a gold mine resource for the development of transgene-based vector control strategies.
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http://dx.doi.org/10.1534/g3.119.401006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056973PMC
March 2020

Development of a confinable gene drive system in the human disease vector .

Elife 2020 01 21;9. Epub 2020 Jan 21.

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

is the principal mosquito vector for many arboviruses that increasingly infect 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 sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in . To fill this void, 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%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of to combat local pathogen transmission.
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http://dx.doi.org/10.7554/eLife.51701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974361PMC
January 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