Background

W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations.

Methods

We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors.

Results

Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR.

Conclusion

Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.

中文翻译：

---

Cas9和Cas12a CRISPR编辑方法校正W1282X-CFTR突变的比较

背景

W1282X-CFTR 变体 (c.3846G>A) 是囊性纤维化跨膜电导调节器 ( CFTR ) 基因中第二常见的无义囊性纤维化 (CF) 突变。尽管在四种 CFTR 蛋白调节剂的批准下，CF 治疗取得了显着的突破，但这些都没有被批准用于具有无义突变的患者。CRISPR基因编辑工具对于永久纠正由这些突变引起的遗传缺陷具有重要价值。

方法

我们使用 Cas9/gRNA 和 Cas12a/gRNA 核糖核蛋白 (RNP) 和单链 DNA (ssODN) 寡核苷酸供体。

结果

Cas9 显示出比 Cas12a 更高的校正水平，因为通过使用 Cas9 RNP 和 ssODN 供体对细胞进行电穿孔，实现了近 18% 的精确编辑，而 Cas12a 仅为 8%。这种校正水平增加了 CFTR mRNA 和蛋白质的丰度，并将编辑细胞池中的 CFTR 功能部分恢复到 WT CFTR 功能的 18%。此外，纯合校正克隆产生的 mRNA、蛋白质和功能水平与表达 WT CFTR 的细胞相当。

结论

总而言之，这项工作证明了基因编辑作为 CF 直接纠正疾病根本原因的治疗策略的潜力。