PDF(Pubmed)
Abstract：
Cas12a (Cpf1), a Class 2 Type V CRISPR/Cas nuclease, has several unique attributes for genome editing and may provide a valuable alternative to Cas9. However, a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application. In this report, we generated two variants, ttAsCas12 Ultra and ttLbCas12a Ultra harboring three (E174R, M537R, and F870L) or two (D156R and E795L) mutations, respectively, by combining the mutations from the temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). We compared editing efficiencies of the five resulting Cas12a variants (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra, and ttAsCas12 Ultra) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). The variant ttLbCas12a Ultra, harboring the D156R and E795L mutations, exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 °C. In addition, optimization of ttLbCas12a Ultra, by varying nuclear localization signal sequences and codon usage, further greatly improved editing efficiency. Collectively, our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00144-w.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00144-w.
摘要：
Cas12a(Cpf1),2类V型CRISPR/Cas核酸酶，具有基因组编辑的几个独特属性，可能为Cas9提供有价值的替代方案。然而,由于温度敏感性而导致的低编辑效率和Cas12a核酸酶的裂解活性不足是其广泛应用的主要障碍。在这份报告中,我们生成了两个变体，ttAsCas12Ultra和ttLbCas12aUltra拥有三个（E174R，M537R,和F870L）或两个（D156R和E795L）突变，分别,通过组合来自温度耐受变体ttAsCas12a（E174R）和ttLbCas12a（D156R）的突变，以及来自高活性变体AsCas12aUltra（M537R和F870L）和LbCas12aUltra（E795L）的那些。我们比较了五种所得Cas12a变体的编辑效率(LbCas12a，ttLbCas12a,ttLbCas12aUltra,AsCas12aUltra,和ttAsCas12Ultra）在拟南芥（拟南芥）中四个基因的六个靶位点。变种ttLbCas12aUltra，有D156R和E795L突变,在拟南芥中测试的所有变体中表现出最高的编辑效率，并且可用于在22°C下生长的拟南芥植物中在单个世代中产生纯合或双等位基因突变体。此外,ttLbCas12aUltra的优化，通过改变核定位信号序列和密码子使用，进一步大大提高了编辑效率。总的来说，我们的结果表明，ttLbCas12aUltra是一个有价值的替代Cas9编辑基因或启动子在拟南芥。
■在线版本包含补充材料，可在10.1007/s42994-024-00144-w获得。
■在线版本包含补充材料，可在10.1007/s42994-024-00144-w获得。
