蚊子人口修改:消除疟疾的动力

Laboratory members from the University of California, Irvine Malaria Initiative, and their collaborators, have been using a prototype Cas9/gRNA-based gene drive system for mosquito population modification with the aim of controlling malaria transmission by targeting the vectors.

世界上78亿人中约有3.4人有感染疟疾寄生虫和发展疾病的风险(世界卫生组织2019年)。在本世纪之交开始了重新努力消除疟疾,在该计划的前15年中,疾病发病率和患病率显着降低。但是,在过去几年中,维持这种势头的努力失败了,剥离的进步已经步履蹒跚。Vector control is a major cost-effective component of the eradication agenda and 80% of the impact on decreasing malaria since 2000 is attributed to anti-vector measures (见1和2)。与这种影响一致WHO recommended research and development新一代的矢量控制工具,以帮助满足根除挑战。

我们的实验室成员和合作者是加利福尼亚大学尔湾疟疾倡议的许多科学家(UCIMI)和塔塔遗传与社会研究所(tigs)seeking to apply genetic engineering technologies to develop novel methods for controlling parasite transmission by targeting the mosquito vectors. Our preferred strategy,人口修改,使用遗传工具使媒介蚊子无法传播寄生虫。我们预计,赋予寄生虫耐药性的基因蚊子种群的引入应导致病原体的传播减少。我们使用合成方法来制造干扰寄生虫传播并通过蚊子种群传播的基因。实施了这种策略后,我们希望看到疟疾传播和疾病的可测量减少。

过去,我们取得了很大的成功,表明我们可以使用现代分子生物学和昆虫转基因工具来制造防止蚊子传播的基因(见1和2)。我们最近专注于实验室实验,以找到将这些基因转移到野生蚊子种群中的方法。

为此,我们和其他人致力于利用CAS9/guide RNA- (Cas9/gRNA) mediated gene drive systems.

图1.种系中活跃的自主基因驱动系统(a)包括编码Cas9核酸酶(橙色)的基因,靶向选择基因的指导RNA编码DNA(黄色)(蓝色)和抗原分子(绿色)。系统切割(剪刀)同源染色体(全蓝色),并通过同源指导的修复(HDR)将其自身从一个染色体复制到其同源物。这会导致遗传性偏见,所有后代都会从该种系细胞收到驱动系统的副本。有时,该系统无法充当裂解后(B)和非同源末端连接(EJ)后的DNA修复模板,可能会引入插入或缺失(红色)。这些突变可能对进一步的CAS9/GRNA裂解具有抵抗力,并表现为抗驱动等位基因,从该种系产生1:1的遗传模式。改编自阿道夫2020

这种聪明而创新的遗传方法促进了从杂合状态到纯合性的基因,等位基因的替代形式的二倍体生物的转化。这项看似简单的技术可以作为一种机制来制造一种基因驱动系统,该系统迅速在靶向蚊子种群中迅速传播抗寄生虫基因。

We demonstrated previouslya prototype Cas9/gRNA-based gene drive system for population modification of the Indo-Pakistan malaria vector,亚线史蒂芬西。该系统是自主的,也就是说,它传达了所有必要的信息以保持自身行驶,并可以移动双重反式抗Plasmodium falciparum(最致命的人类疟原虫)基因占据了约99.5%的后代,后代驱动线向野生型蚊子发出。该驱动器系统的小笼子试验显示,单个释放1:1基因驱动器:野生型男性可以达到100%的引入(每个蚊子至少携带驱动系统和货物的副本)在6-12代内(图2A)。

Figure 2. Comparisons of gene drive system (A-C) and population size (D-F) dynamics in triplicate laboratory cage trials. Drive spread is measured as the change in frequency of dominant fluorescent eye-color phenotypes over multiple, successive, non-overlapping generation (G0-G20 as indicated) following a 1:1 releases of gene-drive system-containing to wild-type males. Note that two of three cages in the prototype gene-drive system cages went extinct after achieving homozygosity (A, D:). The third cage did not achieve homozygosity and maintained a population (A, D:) . Cages with second generation (recoded or alternate gRNA target genes B, E and C, F, respectively, drive systems achieved homozygosity and maintained populations. Abbreviations: DsRed+, Discosoma sp, red fluorescent protein; GFP+, green fluorescent protein; CFP+, cyan fluorescent protein.

Furthermore, we observed that disruption of the target locus for insertion of the drive system,Kynurenine羟化酶((kh,也称为kynurenine单氧酶,KMO,Gene),一种编码在眼睛色素合成途径中重要的酶的基因,在血液中显着影响女性的生存和后代的产生(图3)。

图3.蚊子色素(Ommochrome)生物合成途径中的基因驱动靶标。An的基因驱动靶向。Stephensi kynurenine羟化酶(AskH,虚线)或AN。冈比亚红衣主教(AGCD,虚线蓝线)基因导致成年人在相应的突变等位基因纯合子中可见的眼色表型(图4)。缩写:TDO,色氨酸-2,3-二氧酶;Kat,Kynurenine氨基转移酶;KH,khnurenine羟化酶(KMO,Kynurenine单氧酶);HKT,3-羟基基硝酸转氨酸酶。改编自Carballar-Lejarazú等,2020。

一种方法涉及高效2nd- 原始基因驱动系统的代代版本被指定为“重新编码”,该系统将系统与靶基因的功能副本相吻合(驱动器插入其中)Figure 4)。In contrast to the original system, female mosquitoes of this strain show strong and consistent drive in trials in population cages and negligible production of drive-resistant alleles (Figure 2B, 2E). Mechanistically, non-functional resistant alleles are eliminated via a maternal lethal/sterile effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials showed that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios. This strategy of inserting a gene-drive into an essential gene (for viability or fertility) and then rescuing it with a functional geneprovides a general solutionto the emergence of drive-resistance through females.

Figure 4. Eye-color phenotypes of adult Anopheles gene-drive mosquitoes. Top: wild-type An. stephensi eyes (kh+) mutated to a white-eye phenotype in mosquitoes homozygous for the gene drive system (kh-) show red fluorescent eyes (DsRed+). The same mosquitoes rescued by the Recoding system (khR) have wild-type color eyes marked with green fluorescence (GFP+). Bottom: wild-type An. gambiae eyes (cd+) mutated to a red-eye phenotype in mosquitoes homozygous for the gene drive system (cd-) show cyan fluorescent eyes (CFP+). Abbreviations: kh+, wild-type kynurenine hydroxylase gene; kh-, mutant kynurenine hydroxylase gene; khR, recoded kynurenine hydroxylase gene; cd+ wild-type cardinal gene; cd-, mutant cardinal gene; DsRed+, Discosoma sp, red fluorescent protein; GFP+, green fluorescent protein; CFP+, cyan fluorescent protein. Photograph credits: Kiona Parker, Rebeca Carballar-Lejarazú and Anthony A. James, University of California, Irvine.

第二种方法是将CAS/GRNA靶向基因的靶标改变对适应性没有影响。这个2nd- 为非洲疟疾媒介开发的代理系统,An, gambiae,也针对一个基因,cardinal((cd),还编码参与合成眼颜色色素的酶(Figures 3 and 4)。Thecdgene product acts ‘downstream’ from thekh纯合女性耐受驱动元件插入引起的产物和突变。该驱动器的平均效率在两个性别的平均效率均为约97%体外,几乎没有对驾驶蚊子的适应性以及可能抵抗它的潜在抗性等位基因的低频率。重要的是,实验室中的小笼子中的试验表明,以1:1的比例释放出驾驶雄性的单个释放足以确保每个蚊子在四到六代内至少携带了驱动器构造的至少一个副本,这代表了一个时期6个月,属于单个年度疟疾传输周期(图2C和2F)。该系统可以提供强大的车辆将抗寄生虫效应子基因渡入蚊子中,并可以帮助加快驾驶蚊子菌株的发展,准备进行现场测试。

其他人有效利用了CAS9/GRNA系统的使用(请参阅靶向疟疾伦敦帝国学院的团队)。作为参与本科学发展的大型社区的一部分,我们坚持严格recommendations准则确保安全大调的ing the development of these systems. In addition, all of the work is made available through open access sources (for examplePubMed)允许专家和感兴趣的利益相关者自由访问权限,可以随着进步的发展和评估科学。我们预计这种透明度加上利益相关者的领导将导致该技术的安全且科学上的现场试验。

致谢:此处审查的工作得到了加利福尼亚大学的部分支持尔湾疟疾倡议,塔塔遗传学与社会研究所以及国立卫生研究院(NIH)(AI29746)。A.A.J.是加州大学尔湾分校的唐纳德·布伦教授。

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