單股DNA同源重組提升CRISPR-Cpf1於啤酒酵母菌之基因編輯效率

Enhancement of CRISPR-Cpf1 Genome Editing Efficiency Using ssDNA Homologous Recombination in Saccharomyces cerevisiae

陳柏洲(Bo-Chou Chen) ； 朱文深(Wen-Shen Chu) ； 林奐妤(Huan-Yu Lin)

《臺灣農業化學與食品科學》 59卷1期 (2021/03) Pp. 4-10

https://doi.org/10.6578/TJACFS.202103_59(1).0002

摘要

啤酒酵母菌是人類利用最早且最廣泛，也是可直接食用的微生物之一，在食品工業上的應用已超過數百年歷史，包括烘焙、釀造、製酒等。近年來，透過分子生物技術優化酵母菌，大幅提升酵母菌於食品產業上之應用加值，然而，開發食品工業特殊功能性之酵母菌已越來越嚴苛。透過建立啤酒酵母菌之基因編輯技術，提升基因編輯效率，使其能具備更高之應用價值。本研究以CRISPR-Cpf1系統剔除啤酒酵母菌之ade2基因，分析帶有紅色色素之菌落，證明此系統在啤酒酵母菌進行基因編輯之可行性。在分析三種同源重組模板對於基因編輯之效率，以單股DNA模板進行基因編輯之效率高達67.4％，進一步提升啤酒酵母菌CRISPR-Cpf1基因編輯工具之應用性。

關鍵字

啤酒酵母菌 ； 基因編輯技術 ； CRISPR-Cpf1系統 ； 剔除ade2 ； 視覺辨識菌落 ； 色素

並列摘要

Saccharomyces cerevisiae is one of the most widely used and directly edible microorganisms. It has been used in food industry for more than hundreds of years, including baking, brewing, wine making. In recent years, molecular biotechnology has been used to optimize yeast, greatly enhance the application value of yeast in food industry, and develop special functional yeast. However, the application of yeast in food industry has become and more demanding all over the world. In this study, we choose autotrophy marker gene ade2 as a target gene to knock out by the CRISPR-Cpf1 system in Saccharomyces cerevisiae and used visual selection the pigmented colonies. Then, we examined the genome editing efficiency using three kinds of homologous recombination templates, and the genome editing efficiency of 67.4% was obtained using single-stranded DNA donor templates. Make the CRISPR-Cpf1 genome editing tool in Saccharomyces cerevisiae more applicable.

並列關鍵字

Saccharomyces cerevisiae ； Genome editing technology ； CRISPR-Cpf1system ； Knock-out ade2 ； Visual selection of colonies ； Pigmentation