Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome.

IF 3.7 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS Biological Procedures Online Pub Date : 2023-06-24 DOI:10.1186/s12575-023-00210-5

Nima Dehdilani, Lena Goshayeshi, Sara Yousefi Taemeh, Ahmad Reza Bahrami, Sylvie Rival Gervier, Bertrand Pain, Hesam Dehghani

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引用次数: 1

Abstract

Background: One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome.

Results: Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter.

Conclusions: cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.

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整合组学和CRISPR技术鉴定和验证鸡基因组安全港位点。

背景:转基因领域最突出的问题之一是“基因组中的何处整合转基因?”逃避表观遗传沉默和启动子关闭的转基因需要可靠的基因组安全港(GSH)位点。基因组工程技术的进步与多组学生物信息学数据的结合，使宿主基因组中谷胱甘肽位点的合理评估成为可能。目前，还没有在鸡基因组中鉴定出有效的谷胱甘肽位点。结果:在这里，我们分析和实验检测了鸡细胞基因组中的两个GSH位点。为此，推定的谷胱甘肽基因座包括鸡hip -like (chip;DRG1和EIF4ENIF1基因之间的差异)和鸡rosa样(cROSA;使用多组学生物信息学数据预测了THUMPD3基因上游的基因。然后，通过对连续培养的同源细胞克隆进行实验表征，验证了转基因的持久表达，该克隆在预测位点中含有DsRed2-ΔCMV-EGFP盒。与全长CMV启动子相比，CMV启动子的弱化形式(ΔCMV)允许以位点依赖的方式精确评估GSH位点。结论:本研究中引入的chip和cROSA基因座可以可靠地用于基因工程鸡中重组蛋白的一致性生物制造。此外，研究结果还表明，基因组背景决定了由ΔCMV控制的转基因基因在GSH位点的表达。

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来源期刊

Biological Procedures Online 生物-生化研究方法

CiteScore

10.50

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： iological Procedures Online publishes articles that improve access to techniques and methods in the medical and biological sciences. We are also interested in short but important research discoveries, such as new animal disease models. Topics of interest include, but are not limited to: Reports of new research techniques and applications of existing techniques Technical analyses of research techniques and published reports Validity analyses of research methods and approaches to judging the validity of research reports Application of common research methods Reviews of existing techniques Novel/important product information Biological Procedures Online places emphasis on multidisciplinary approaches that integrate methodologies from medicine, biology, chemistry, imaging, engineering, bioinformatics, computer science, and systems analysis.