Cebrian-Serrano Alberto, Davies Benjamin
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
Mamm Genome. 2017 Aug;28(7-8):247-261. doi: 10.1007/s00335-017-9697-4. Epub 2017 Jun 20.
Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases.
由于CRISPR-Cas系统中RNA引导的核酸内切酶的发现，现在在各种细胞和模式生物中进行强大且具有成本效益的基因组编辑成为可能。化脓性链球菌常用的Cas9显示出高水平的活性，但根据应用情况，也存在一些缺点。首先，该酶已被证明会在类似于靶序列的基因组序列处引起诱变。其次，对所选靶位点相邻的特定基序的严格要求会限制这种酶的靶标范围。最后，Cas9的物理尺寸对基于该酶作为潜在治疗应用的病毒颗粒的基因组工程工具的有效递送提出了挑战。已经采用了相关和并行的策略来解决这些问题。利用可获得的大量关于CRISPR-Cas效应蛋白的结构信息，通过诱变对活性和靶标识别有贡献的关键残基对Cas9进行了重新设计。该蛋白也被缩短并重新设计成组成亚基，以促进其有效递送。此外，通过探索来自不同细菌物种的Cas9直系同源物和其他相关效应蛋白的特性，扩展了CRISPR-Cas工具箱，其中一些表现出不同的靶位点特异性和减小的分子尺寸。希望在准确性、靶标范围和递送效率方面的改进将促进这些位点特异性核酸酶的治疗应用。
