Efficient CRISPR-Cas9 editing of major evolutionary loci in sticklebacks.

Q2 Agricultural and Biological Sciences
Evolutionary Ecology Research Pub Date : 2019-01-01
Julia I Wucherpfennig, Craig T Miller, David M Kingsley
{"title":"Efficient CRISPR-Cas9 editing of major evolutionary loci in sticklebacks.","authors":"Julia I Wucherpfennig,&nbsp;Craig T Miller,&nbsp;David M Kingsley","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Stickleback fish are widely used to study the genetic and ecological basis of phenotypic evolution. Although several major loci have now been identified that contribute to evolutionary differences between wild populations, further study of the phenotypes associated with particular genes and mutations has been limited by the difficulty of generating targeted mutations at precise locations in the stickleback genome.</p><p><strong>Approach and aims: </strong>We compared different methods of expressing single-guide RNAs (sgRNAs) and Cas9 activity in fertilized stickleback eggs. We used an easily scored pigmentation gene (<i>SLC24A5</i>) to screen for molecular lesions, phenotypic effects, and possible germline transmission of newly induced alleles. We then used the optimized CRISPR methods to target two major evolutionary loci in sticklebacks, <i>KITLG</i> and <i>EDA</i>. We hypothesized that coding region mutations in the <i>KITLG</i> gene would alter body pigmentation and possibly sex determination, and that mutations in the <i>EDA</i> gene would disrupt the formation of most armor plates, fin rays, spines, teeth, and gill rakers.</p><p><strong>Results: </strong>Targeted deletions were successfully induced at each target locus by co-injecting one-cell stage stickleback embryos with either <i>Cas9</i> mRNA or Cas9 protein, together with sgRNAs designed to protein-coding exons. Founder animals were typically mosaic for multiple mutations, which they transmitted through the germline at overall rates of 21 to 100%. We found that the copy of <i>KITLG</i> on the X chromosome (<i>KITLGX</i>) has diverged from the <i>KITLG</i> on the Y chromosome (<i>KITLGY</i>). Predicted loss-of-function mutations in the <i>KITLGX</i> gene dramatically altered pigmentation in both external skin and internal organ, but the same was not true for <i>KITLGY</i> mutations. Predicted loss-of-function mutations in either the <i>KITLGX</i> or <i>KITLGY</i> genes did not lead to sex reversal or prevent fertility. Homozygous loss-of-function mutations in the <i>EDA</i> gene led to complete loss of armor plates, severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in tooth and gill raker number. In contrast, long dorsal and pelvic spines remained intact in <i>EDA</i> mutant animals, suggesting that common co-segregation of plate loss and spine reduction in wild populations is unlikely to be due to pleiotropic effects of <i>EDA</i> mutations.</p><p><strong>Conclusion: </strong>CRISPR-Cas9 approaches can be used to induce germline mutations in key evolutionary loci in sticklebacks. Targeted coding region mutations confirm an important role for <i>KITLG</i> and <i>EDA</i> in skin pigmentation and armor plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.</p>","PeriodicalId":50469,"journal":{"name":"Evolutionary Ecology Research","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8664273/pdf/nihms-1667662.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Evolutionary Ecology Research","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}
引用次数: 0

Abstract

Background: Stickleback fish are widely used to study the genetic and ecological basis of phenotypic evolution. Although several major loci have now been identified that contribute to evolutionary differences between wild populations, further study of the phenotypes associated with particular genes and mutations has been limited by the difficulty of generating targeted mutations at precise locations in the stickleback genome.

Approach and aims: We compared different methods of expressing single-guide RNAs (sgRNAs) and Cas9 activity in fertilized stickleback eggs. We used an easily scored pigmentation gene (SLC24A5) to screen for molecular lesions, phenotypic effects, and possible germline transmission of newly induced alleles. We then used the optimized CRISPR methods to target two major evolutionary loci in sticklebacks, KITLG and EDA. We hypothesized that coding region mutations in the KITLG gene would alter body pigmentation and possibly sex determination, and that mutations in the EDA gene would disrupt the formation of most armor plates, fin rays, spines, teeth, and gill rakers.

Results: Targeted deletions were successfully induced at each target locus by co-injecting one-cell stage stickleback embryos with either Cas9 mRNA or Cas9 protein, together with sgRNAs designed to protein-coding exons. Founder animals were typically mosaic for multiple mutations, which they transmitted through the germline at overall rates of 21 to 100%. We found that the copy of KITLG on the X chromosome (KITLGX) has diverged from the KITLG on the Y chromosome (KITLGY). Predicted loss-of-function mutations in the KITLGX gene dramatically altered pigmentation in both external skin and internal organ, but the same was not true for KITLGY mutations. Predicted loss-of-function mutations in either the KITLGX or KITLGY genes did not lead to sex reversal or prevent fertility. Homozygous loss-of-function mutations in the EDA gene led to complete loss of armor plates, severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in tooth and gill raker number. In contrast, long dorsal and pelvic spines remained intact in EDA mutant animals, suggesting that common co-segregation of plate loss and spine reduction in wild populations is unlikely to be due to pleiotropic effects of EDA mutations.

Conclusion: CRISPR-Cas9 approaches can be used to induce germline mutations in key evolutionary loci in sticklebacks. Targeted coding region mutations confirm an important role for KITLG and EDA in skin pigmentation and armor plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.

Abstract Image

Abstract Image

Abstract Image

棘鱼主要进化位点的高效CRISPR-Cas9编辑。
背景:棘鱼被广泛用于研究表型进化的遗传和生态基础。虽然现在已经确定了几个主要的基因座对野生种群之间的进化差异有贡献,但由于在棘鱼基因组的精确位置产生靶向突变的困难,与特定基因和突变相关的表型的进一步研究受到了限制。方法与目的:我们比较了棘鱼受精卵中表达单导rna (single-guide RNAs, sgRNAs)和Cas9活性的不同方法。我们使用一个易于标记的色素沉着基因(SLC24A5)来筛选新诱导等位基因的分子病变、表型效应和可能的种系传播。然后,我们使用优化的CRISPR方法针对刺鱼的两个主要进化位点KITLG和EDA。我们假设KITLG基因的编码区突变会改变身体的色素沉积,并可能改变性别决定,而EDA基因的突变会破坏大多数甲片、鳍、刺、牙齿和鳃柄的形成。结果:通过共注射含有Cas9 mRNA或Cas9蛋白的单细胞期棘鱼胚胎,以及设计为蛋白质编码外显子的sgRNAs,成功地在每个目标位点诱导了靶向缺失。创始者的动物通常是多种突变的嵌合体,它们通过种系传播的总体比率为21%至100%。我们发现X染色体上的KITLG拷贝(KITLGX)与Y染色体上的KITLG拷贝(KITLGY)发生了分化。KITLGX基因预测的功能缺失突变显著改变了外部皮肤和内部器官的色素沉着,但KITLGX基因突变并非如此。KITLGX或KITLGY基因中预测的功能丧失突变不会导致性别逆转或阻止生育。EDA基因的纯合子功能缺失突变导致甲片完全丧失,背鳍、肛门鳍和尾鳍中大多数软射线严重减少或丧失,牙齿和鳃柄数量严重减少。相比之下,EDA突变动物的长背和骨盆脊柱保持完整,这表明野生种群中钢板丢失和脊柱减少的共同分离不太可能是由于EDA突变的多益性作用。结论:CRISPR-Cas9方法可用于诱导棘鱼关键进化位点的种系突变。靶向编码区突变证实了KITLG和EDA分别在皮肤色素沉着和盔甲减少中发挥重要作用。它们还提供了关于这些基因在其他身体结构中的功能的新信息。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Evolutionary Ecology Research
Evolutionary Ecology Research 生物-进化生物学
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: Evolutionary Ecology Research publishes original research contributions focusing on the overlap between ecology and evolution. Papers may treat any taxon or be general. They may be empirical, theoretical or a combination of the two. EER prefers conceptual contributions that take intellectual risks or that test ideas.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信