The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system

IF 3.1 2区 生物学 Q2 ECOLOGY
Sen Xu, Swatantra Neupane, Hongjun Wang, Thinh Phu Pham, Marelize Snyman, Trung V. Huynh, Li Wang
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Abstract

The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases. Our data suggest site-specific DNA cleavage mostly occurs in a stepwise fashion. Indels dominate the induced mutations. A few, unexpected on-site large deletions (>1 kb) are also observed. Notably, genome-wide analyses reveal no off-target mutations. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progeny. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes involved in human neurodegenerative diseases.

Abstract Image

在一个新兴的模式系统中cas诱导突变的镶嵌和猩红基因的多效效应。
CRISPR技术在新兴模式生物中的有效应用面临着有效产生可遗传突变和理解诱导DNA损伤的基因组后果和诱导突变的遗传模式的重大挑战。本研究解决了这些问题,1)开发了一种高效的微注射递送方法,用于微甲壳类动物水蚤的基因编辑;2)评估Cas9和Cas12a核酸酶在猩红基因敲除突变体中的编辑动态;3)研究猩红突变体的转录组,了解猩红基因的多效性。我们重新设计的显微注射方法可以有效地编辑两种核酸酶的双等位基因。我们的数据表明,位点特异性DNA切割主要以逐步的方式发生。indel主导诱导突变。还观察到一些意想不到的现场大缺失(bbb1kb)。值得注意的是,全基因组分析显示没有脱靶突变。尽管靶位点周围有复杂的诱导突变,但将终止密码子盒敲入到猩红位点是成功的。此外,在一些突变体中存在广泛的种系嵌合现象,这些突变体在其无性后代中意外地产生不同的表型/基因型。最后,我们的转录组学分析揭示了突变体中与猩红基因敲除和游泳行为改变相关的显著基因表达变化,包括与人类神经退行性疾病有关的几个基因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Heredity
Heredity 生物-进化生物学
CiteScore
7.50
自引率
2.60%
发文量
84
审稿时长
4-8 weeks
期刊介绍: Heredity is the official journal of the Genetics Society. It covers a broad range of topics within the field of genetics and therefore papers must address conceptual or applied issues of interest to the journal''s wide readership
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