Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.
Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.
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DOI：
10.1093/nar/gkad1125
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发表时间：
2024-01-25
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
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作者：
向导RNA为CRISPR-Cas9基因组编辑提供了可编程性，但也增加了递送的挑战。化学修饰是寡核苷酸治疗成功的关键，可以增强核酸的稳定性、分布、细胞摄取和安全性。之前，我们设计了高度和完全修饰的SpyCas 9 crRNA和tracrRNA，当以核糖核蛋白复合物的形式递送到培养的细胞时，其显示出增强的稳定性和保留的活性。在这项研究中，我们报告了一种短的、完全稳定的寡核苷酸（一种“保护性寡核苷酸”），它可以通过tracrRNA退火来取代，可以显着增强重度修饰的crRNA的效力和稳定性。此外，保护性寡核苷酸允许附加各种生物缀合物，从而改善crRNA在体内的细胞摄取和生物分布。最后，我们通过共递送未配制的化学修饰的crRNA与表达tracrRNA和SpyCas 9或碱基编辑衍生物的保护性寡核苷酸和AAV载体，在成年小鼠肝脏和中枢神经系统中实现了体内基因组编辑。我们的AAV/crRNA共递送的概念验证建立提供了通向瞬时编辑活性、靶多重化、引导再给药和载体失活的途径。
Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a ‘protecting oligo’), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.
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DOI：
10.1016/j.ymthe.2020.12.023
发表时间：
2021-04-07
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
--
作者：
Biscans A;Caiazzi J;McHugh N;Hariharan V;Muhuri M;Khvorova A
通讯作者：
Khvorova A
影响因子：
64.8
作者：
Anzalone, Andrew V.;Randolph, Peyton B.;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
5.3
作者：
Bon, Charlotte;Hofer, Thomas;Krippendorff, Ben-Fillippo
通讯作者：
Krippendorff, Ben-Fillippo
影响因子：
14.9
作者：
Biscans, Annabelle;Coles, Andrew;Khvorova, Anastasia
通讯作者：
Khvorova, Anastasia
影响因子：
8.8
作者：
Allison, Simon J.;Milner, Jo
通讯作者：
Milner, Jo