清脆的
电穿孔
脂质体
Cas9
合子
基因组编辑
生物
转染
胚胎
外源DNA
遗传增强
细胞生物学
分子生物学
基因
遗传学
胚胎发生
重组DNA
载体(分子生物学)
作者
Koki Takebayashi,Manita Wittayarat,Qingyi Lin,Maki Hirata,Naoaki Yoshimura,Nanaka Torigoe,Megumi Nagahara,Lanh Thi Kim,Fuminori Tanihara,Takeshige Otoi
摘要
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.
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