Precise therapeutic gene correction by a simple nuclease-induced double-stranded breaky
Precise therapeutic gene correction by a simple nuclease-induced double-stranded breaky
复制标题
DOI：
10.1038/s41586-019-1076-8
复制
发表时间：
2019-04-25
期刊：
影响因子：
64.8
通讯作者：
Wolfe, Scot A.
中科院分区：
文献类型：
--
作者：
Iyer, Sukanya;Suresh, Sneha;Wolfe, Scot A.
目前用于精确校正致病基因突变的基于可编程核酸酶的方法（例如CRISPR-Cas9）利用同源定向修复途径。然而，这种修复过程需要共同递送外源DNA供体以重新编码序列，并且在许多细胞类型中可能是低效的。在这里，我们表明，致病移码突变，导致微复制可以有效地恢复到野生型序列，只需在复制中心附近产生一个DNA双链断裂。我们在两种疾病的患者来源的细胞系中证明了这一点：肢带型肌营养不良症2G型（LGMD 2G）（1）和Hermansky-Pudlak综合征1型（HPS 1）（2）。来自LGMD 2G细胞系的诱导型多能干（iPS）细胞的克隆分析，其含有TCAP突变，用化脓链球菌Cas9（SpCas 9）核酸酶处理，显示约80%含有至少一个野生型TCAP等位基因;这种校正也恢复了LGMD 2G iPS细胞衍生的肌管中的TCAP表达。SpCas 9还有效地校正了HPS 1患者来源的B淋巴母细胞样细胞系的基因型。抑制聚ADP-核糖聚合酶1（PARP-1）抑制了核酸酶介导的微复制到野生型序列的崩溃，证实了精确的校正是由微同源介导的末端连接（MMEJ）途径介导的。SpCas 9和毛螺菌科细菌ND 2006 Cas 12 a（LbCas 12 a）在基因组内的非致病性4-36-碱基对微重复处的编辑分析表明，校正策略广泛适用于广泛的微重复长度，并且可以由多种核酸酶启动。这种基于MMEJ的治疗策略的简单性、可靠性和有效性应该允许开发用于与微复制相关的各种疾病的基于核酸酶的基因校正疗法。
Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wildtype sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)(1) and Hermansky-Pudlak syndrome type 1(HPS1)(2). Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wildtype sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.