基因君官网
2016年1月11日 讯 /生物谷BIOON/ –一种编辑基因组的强大技术或将具有更高地精确性，6日发表于国际杂志Nature上的研究报告中，研究人员巧妙地利用一种酶类，成功地降低了在某些情况下CRISPR–Cas9技术的错误率。
研究人员利用CRISPR–Cas9技术可以对基因组进行精确修饰来移除或者编辑错误的基因，而且该技术可以对包括人类胚胎在内的任何生物进行编辑；CRISPR–Cas9技术依赖于酶类Cas9，其可以利用一种导向RNA分子安全返回到自身的靶向DNA上。Cas9分子可以在位点对DNA进行切割，随后细胞中天然的DNA修复机器就会开始修复切口，即剔除短片段的DNA分子或缝合某些序列。
但当前这种技术并不是完全可靠的，有时候Cas9酶会产生未知的突变，目前CRISPR技术已经从实验室转向了临床中，但是否其可以应用于胚胎研究中还存在一定争议；来自MIT的研究者Keith Joung说道，我们希望通过深层次的研究来减少CRISPR–Cas9技术脱靶的可能性；有些研究人员表示，CRISPR技术在临床中使用的错误率并不会减少到零，而有时候每个人都需要决定怎么样的特殊性才足够特殊，而目前开发一种完全没有脱靶可能性的工具似乎有点理想化。
此前研究中，研究人员利用短链的导向RNA指导酶类Cas9直接作用靶向DNA，从而就可以切断某些错误，今年12月生物学家张峰就和同事宣布他们对Cas9进行了工程化修饰使其错误率更低；最新的研究中，研究者Joung和其同事对酶类Cas9的不同区域进行了处理，改变了其同DNA靶点接触的蛋白部分，随后研究者利用更灵敏的方法进行了错误的检测。研究人员利用8个不同的导向RNAs检测了这种名为SpCas9-HF1的高保真酶，这种工程化酶类可以在不改变形态的情况下对靶向DNA进行切割，在一个导向RNAs仅会制造一处错误，相比较而言，没有修饰改变的Cas9酶当被8个RNAs中的7个进行引导时就会产生错误。
Cas9的错误是目前基因组编辑研究领域的一个重要的问题，其中就包括是否该技术可以在人类胚胎中进行应用，研究者Church希望对导向RNA进行精确地涉及从而尽可能避免脱靶缺口的出现。尽管当前的研究工作已经将CRISPR–Cas9技术推向了临床中，但该系统仍需要进行额外的安全性检查才能够应用于人类疾病的治疗中。
12月，研究者Gersbach及其同事宣布他们已经利用CRISPR–Cas9技术成功修复了杜氏肌营养不良症小鼠的遗传突变，为了完成这一目标，研究人员利用一种病毒来将Cas9酶运输到肌肉细胞中，相比研究者Joung的实验而言，研究者Gersbach就可以使得该病毒持续表达酶类，从而大大降低了脱靶缺口的产生。
目前FDA并没有批准在临床试验中应用CRISPR–Cas9技术，但来自加州的圣加莫生物科技公司（Sangamo Biosciences）已经利用了另外一种名为锌指核酸酶的基因编辑工具对80多名患者成功进行了临床试验。基于当前的临床试验，监管部门需要知道细胞经过修饰后所表现的安全数据以及脱靶突变的相关信息，圣加莫生物科技公司则需要展示出他们修改后的免疫T细胞仍然可以像正常T细胞一样发挥作用，比如修饰后的肝脏细胞仍然可以在不表现出任何毒性的情况下维持自己的功能。
最后研究者Urnov说道，这些研究对Cas9领域的研究带来了巨大的推进，但如今考虑将该基因编辑技术应用于临床领域的话，我们或许还有一段漫长的路要走。（基因宝jiyinbao.com）
本文系生物谷原创编译整理。欢迎转载！转载请 获取授权 。更多资讯请下载生物谷资讯APP。
doi:10.1038/nature16526
PMC:
PMID:
High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects
Benjamin P. Kleinstiver, Vikram Pattanayak, Michelle S. Prew, Shengdar Q. Tsai, Nhu T. Nguyen, Zongli Zheng & J. Keith Joung
CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
Christopher E. Nelson1,2, Chady H. Hakim3, David G. Ousterout1,2, Pratiksha I. Thakore1,2, Eirik A. Moreb1,2, Ruth M. Castellanos Rivera4, Sarina Madhavan1,2, Xiufang Pan3, F. Ann Ran5,6, Winston X. Yan5,7,8, Aravind Asokan4, Feng Zhang5,9,10,11, Dongsheng Duan3,12, Charles A. Gersbach1,2,13,*
Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR/Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR/Cas9-based genome editing as a potential therapy to treat DMD.
Rationally engineered Cas9 nucleases with improved specificity
Ian M. Slaymaker1,2,3,4,*, Linyi Gao1,4,*, Bernd Zetsche1,2,3,4, David A. Scott1,2,3,4, Winston X. Yan1,5,6, Feng Zhang1,2,3,4,†
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.