使用金黄色葡萄球菌Cas9进行体内基因组编辑。
In vivo genome editing using Staphylococcus aureus Cas9.
作者信息
Ran F Ann, Cong Le, Yan Winston X, Scott David A, Gootenberg Jonathan S, Kriz Andrea J, Zetsche Bernd, Shalem Ophir, Wu Xuebing, Makarova Kira S, Koonin Eugene V, Sharp Phillip A, Zhang Feng
机构信息
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Society of Fellows, Harvard University, Cambridge, Massachusetts 02138, USA.
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
出版信息
Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1.
The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.
RNA 引导的核酸内切酶 Cas9 已成为一种多功能的基因组编辑平台。然而，来自化脓性链球菌（SpCas9）的常用 Cas9 的大小限制了其在基础研究和使用高度通用的腺相关病毒（AAV）递送载体的治疗应用中的效用。在此，我们对六个较小的 Cas9 直系同源物进行了表征，并表明来自金黄色葡萄球菌的 Cas9（SaCas9）能够以与 SpCas9 相似的效率编辑基因组，同时其长度比 SpCas9 短 1 千碱基以上。我们将 SaCas9 及其单向导 RNA 表达盒包装到单个 AAV 载体中，并在小鼠肝脏中靶向胆固醇调节基因 Pcsk9。在注射后的一周内，我们观察到基因修饰率超过 40%，同时血清 Pcsk9 和总胆固醇水平显著降低。我们使用 BLESS 进一步评估了 SaCas9 和 SpCas9 的全基因组靶向特异性，并证明 SaCas9 介导的体内基因组编辑具有高效和特异性的潜力。
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