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The Effect of GC Content in Efficiency of CRISPR/Cas9 Gene Targeting Technology
CRISPR-Cas9,knockout efficiency,GC content,segment design,
|Publication Year :||2017|
|Abstract:||Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9)自從2012年由細菌免疫系統發展為基因標靶 (gene targeting) 工具後，因其便利及高效性，迅速取代鋅指蛋白核酸酶 (zinc-finger nuclease, ZFN) 及transcription activator-like effector nuclease (TALEN)。CRISPR-Cas9系統含sgRNA (single guide RNA) 與Cas9兩功能角色，sgRNA為短片段單股RNA，3’端之二級結構可與Cas9結合，以5’端之20個鹼基辨識DNA序列，最後藉Cas9之核酸酶活性對目標DNA截切造成雙股斷裂 (double strand break)。先前研究發現不同目標DNA序列對CRISPR-Cas9作用效率造成大幅度波動，介於0-100%，如何維持高作用效率為目前待解決之問題。GC含量為DNA序列基本特徵，研究顯示其亦為影響CRISPR-Cas9作用效率之重要因子，當目標DNA序列之GC含量低於30%或高於80%會抑制其作用效率，且發現高於50%時有較高效率，有潛力可作為挑選目標序列之依據。本研究根據CRISPR-Cas9作用機制對sgRNA序列辨識區進行分段設計，比較3’端 (第1-6個鹼基)、中間區段 (第7-12個鹼基) 及5’端 (第13-20個鹼基)，深入探討GC含量對作用效率之影響。試驗首先針對EGFP DNA序列設計22條sgRNA，以EGFP轉基因小鼠之胚胎纖維母細胞 (mouse embryonic fibroblast, MEF) 進行體外試驗，將表現質體以脂質體轉染 (liposome transfection, lipofection) 轉入MEF，測試不同sgRNA對EGFP剔除作用效率。此後根據細胞試驗結果，挑選4條sgRNA以小鼠胚進行體內試驗，將體外轉錄 (in vitro transcription, IVT) 製備之RNA對小鼠胚細胞質進行顯微注射 (microinjection)，此後將胚培養至囊胚或移入代理孕母，計算囊胚及出生小鼠EGFP剔除效率，並與MEF之體外試驗結果比較。試驗結果顯示sgRNA GC含量提高時EGFP剔除作用效率有下降趨勢，GC含量50%以下作用效率較高，此外降低sgRNA中間區段GC含量對於作用效率提高效果顯著高於3’端及5’端。根據以上結果，未來可於GC含量較低之DNA序列設計sgRNA，以提高CRISPR-Cas9作用效率。然而，本試驗結果顯示GC含量50%以下呈現較高效率，與先前研究不符，由於目前尚未完全瞭解其分子機制，欲釐清需更深入之探討。|
The robust gene editing tool, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9), was derived from bacteria immune system in 2012. Because of its convenience and high efficiency, it had quickly replaced current gene targeting tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). CRISPR-Cas9 system contains two functional members, one is a short single strand RNA called single guide RNA (sgRNA) and another is the Cas9 nuclease. Cas9 will combine with sgRNA via binding the secondary structure at 3’ end of sgRNA, makes Cas9 transform into an active conformation. And then uses a 20 nucleotide sequence recognized region at 3’ end of sgRNA to recognize target DNA sequence, induces a DNA double strand breaking. The previous study found CRISPR-Cas9 efficiency had a great variation from 0% to 100% while targeting different target sequence. Made it necessary to figure out how to find a high-efficiency target sequence. GC content is one of the important DNA characters. Recent reports recommend that CRISPR-Cas9 target site should be 30-80% of GC content to avoid very low efficiency. Besides, GC content higher than 50% is more efficient. These both implied GC content might be a useful parameter to select target DNA region. Therefore, this study focused on how GC content influences CRISPR-Cas9 efficiency. Segmentally designed the sgRNA recognition region and compared the influence of the GC content at 3’ end (1-6 nucleotide), mid region (7-12 nucleotide) and 5’ end (13-20 nucleotide). In this study, we took advantage of the EGFP transgenic mice as the cell and embryo origin and compared the efficiency of 22 sgRNAs targeting EGFP. In the first part, we isolated mouse embryonic fibroblast (MEF) for cell-based in vitro experiment. Transfected the CRISPR-Cas9 expression plasmid into MEF by liposome (lipofection), and compared the EGFP knockout efficiency of 22 sgRNAs. And then, selected 4 sgRNAs to the second part embryo-based in vivo experiment according to cell-based data. Used microinjection method to inject CRISPR RNA into the mouse embryo. Cultured the injected embryo to blastocyst or transfer the embryo to the surrogate female mouse. Calculated EGFP knockout efficiency in blastocyst or pup, and compared the result with cell-based data. The results showed that increasing GC content had a negative effect on EGFP knockout efficiency. sgRNA with GC content lower than 50% had the better EGFP knockout rate. And decreasing GC content at the mid region of sgRNA could enhance EGFP knockout efficiency more than at 3’ or 5’ end. To sum up, in the future, we can design sgRNAthat targeting the DNA sequence with lower GC content to increase the CRISPR-Cas9 efficiency. However, the result that sgRNA with GC content lower than 50% is more efficient is inconsistent. It will need further research to investigate.
|Appears in Collections:||動物科學技術學系|
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