Abstract：
Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR\'s application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.
摘要：
尽管CRISPR-Cas9基因疗法已被证明是许多应用中的强大工具，改进是必要的，以增加该技术的特异性。Cas9切断脱靶位点仍然是一个限制CRISPR在人类治疗中应用的问题。当突变等位基因与野生型序列仅相差一个碱基对时,常染色体显性疾病的治疗也仍然是一个挑战。这里,我们利用合成肽核酸(PNA),其结合引导RNA(gRNA)中的选定间隔序列以将Cas9特异性提高10倍。我们询问PNA长度的变化，结合位置,以及与gRNA的同源性程度。我们的发现表明，PNA结合在远离前间隔区相邻基序（PAM）位点的区域中，可以有效地增强中靶/脱靶和等位基因特异性情况下的特异性。此外,我们证明,在结合在gRNA的PAM近端区域中的PNA之间引入故意的错配可以以等位基因特异性方式调节Cas9活性。这些进步有望解决当前的局限性并扩大CRISPR技术的治疗潜力。
