CRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations.

CRISPR/Cas9是一种很有前景的基因校正技术。然而，该版本通常是双等位基因的，并且会创建伴随精确校正的不受控制的小插入和缺失（indels）。最近测试了突变特异性引导RNA，以纠正显性遗传性疾病，从而避免野生型等位基因的存在。我们测试了纠正复合杂合隐性突变的原始方法。我们比较了来自携带复合杂合突变（导致UROS基因失效）的先天性红细胞生成性卟啉症患者的 iPSC 中双等位基因指导 RNA 与突变等位基因特异性指导 RNA 的编辑效率和基因毒性。我们通过两个指南获得了 UROS 功能挽救和代谢校正，具有用于卟啉症临床干预的潜力。然而，与双等位基因不同的是，突变等位基因特异性指南没有针对目标的附带损害。我们推荐这种设计，以避免遗传毒性，并针对经常出现复合杂合突变的隐性疾病获得靶向无疤痕基因校正。