Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both and . We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency , supporting the use of the X cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome.

中文翻译：

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Cas9 表达的时间限制提高了 CRISPR 介导的缺失效率和保真度

CRISPR-Cas9技术在体细胞突变大量缺失方面的临床应用效率低下，而提高实用性的方法因我们无法快速评估单等位基因缺失和双等位基因缺失而受到影响。在这里，我们建立了一个模型系统，用于在单细胞水平上研究等位基因异质性，并识别 gRNA 切割位点非同时核酸酶活性造成的插入缺失疤痕，这是 CRISPR-del 功效的主要障碍。我们发现，通过诱导型腺相关病毒 (AAV) 或脂质纳米颗粒 (LNP) 限制 CRISPR-Cas9 表达，可以部分阻止非同时核酸酶活性。基于诱导型 AAV 的 CRISPR-del 机制表达显着提高了单等位基因和双等位基因的缺失频率，支持使用 X 盒而不是传统的组成型表达 AAV 方法。这些数据描述了 CRISPR-del 后删除的改进和对等位基因异质性的洞察，将为需要大量单等位基因或双等位基因删除的表型的治疗方法提供信息，例如常染色体隐性遗传疾病或突变等位基因特异性 gRNA 不易获得的情况，或在用于切除的目标序列在基因组中多次出现的情况。