A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation.
A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation.
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DOI：
10.1016/j.omtm.2021.05.014
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发表时间：
2021-09-10
期刊：
影响因子：
--
通讯作者：
Olson EN
中科院分区：
文献类型：
--
作者：
Zhang Y;Nishiyama T;Li H;Huang J;Atmanli A;Sanchez-Ortiz E;Wang Z;Mireault AA;Mammen PPA;Bassel-Duby R;Olson EN
Duchenne肌营养不良症(DMD)是一种致命的神经肌肉疾病，由X连锁dystrophin基因突变引起。通过使用腺相关病毒(AAV)携带的化脓性链球菌Cas9(SpCas9)对CRISPR/Cas9基因组进行编辑，纠正了动物模型中的DMD突变。然而，由于AAV的病毒包装能力有限，需要两个AAV载体来运送SpCas9核酸酶及其单引导RNA(SgRNA)，阻碍了其治疗应用。我们设计了一种高效的单切基因编辑方法，使用致密的金黄色葡萄球菌Cas9(SaCas9)来恢复DMD中最常见的框外显子51的开放阅读框架。人类诱导多能干细胞来源的心肌细胞外显子51的编辑显示出强烈的偏好通过两个核苷酸的缺失来重组外显子。我们采用该系统从单个AAV9载体中表达了SaCas9和sgRNA。在外显子50缺失的DMD小鼠模型中，系统地传递这种一体化AAV9系统恢复了Dstrophin的表达并改善了肌肉收缩能力。这些发现证明了由整合的AAV递送系统提供的CRISPR/SaCas9在体内纠正DMD的有效性，代表了一种很有前途的治疗方法来纠正DMD的遗传原因。CRISPR/Cas介导的基因编辑正被用于治疗目的。在这项研究中，张等人。开发了一种整合的AAV递送系统，将SaCas9核酸酶和sgRNA包装在单个AAV载体中，并纠正人类心肌细胞和小鼠的Duchenne肌营养不良突变。
Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD. CRISPR/Cas-mediated gene editing is being used for therapeutic purposes. In this study, Zhang et al. developed a consolidated AAV delivery system to package both SaCas9 nuclease and sgRNA in a single AAV vector and correct Duchenne muscular dystrophy mutations in human cardiomyocytes and mice.
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