Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions
Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions
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DOI：
10.1016/j.isci.2023.106399
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发表时间：
2023-03-23
期刊：
影响因子：
5.8
通讯作者：
Anegon, Ignacio
中科院分区：
文献类型：
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作者：
Chenouard, Vanessa;Leray, Isabelle;Anegon, Ignacio
CRISPR-Cas9 切割效率和准确性是基因编辑面临的主要挑战，尤其受到核糖核蛋白 (RNP) 复合物最佳形成的影响。我们使用纳米差示扫描荧光测定法（一种无标记和无固定化测定）来证明 Cas9 和引导 RNA (gRNA) 的等摩尔比对于 RNP 复合物的形成是最佳的。当使用 0.4 mM Cas9、等摩尔 Cas9/gRNA 比率和 2 mM 单链寡核苷酸时，我们使用双等位纯合 GFP 人诱导多能干细胞系几乎实现了 50% 的绿色荧光蛋白 (GFP) 到蓝色荧光蛋白 (BFP) 的转化，结果表明增加 Cas9/gRNA 比率并没有进一步提高 KI 效率。此外，过量的 gRNA 降低了大鼠胚胎中点突变 KI 的效率，并大大增加了靶向大缺失的发生率。这些发现强调了 CRISPR/Cas9 化学计量优化对于确保高效、准确的 KI 生成的重要性，这将适用于其他体外和体内模型。
CRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 mM of Cas9, equimolar Cas9/gRNA ratio and 2 mM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models.