Arthropod Genetics, The Pirbright Institute, Ash Road, Woking, Surrey, United Kingdom; Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Curr Opin Biotechnol. 2019 Feb;55:68-73. doi: 10.1016/j.copbio.2018.07.005. Epub 2018 Sep 3.
CRISPR/Cas technologies have rapidly become in routine use for site-directed genetic or transcriptional manipulation. Despite this, the efficiency of CRISPR/Cas9 functioning cannot entirely be predicted, and it is not fully understood which factors contribute to this variability. Recent studies indicate that heterochromatin can negatively affect Cas9 binding and functioning. Investigating chromatin factors indicates that DNA cytosine-5 methylation does not directly block Cas9 binding. Nucleosomes, however, can completely block Cas9 access to DNA in cell-free assays and present a substantial hurdle in vivo. In addition to being associated with an open chromatin state, active transcription can directly stimulate DNA cleavage by influencing Cas9 release rates in a strand-specific manner. With these insights and a better understanding of genome-wide chromatin and transcription states, CRISPR/Cas9 effectiveness and reliability can be improved.
CRISPR/Cas 技术已迅速成为用于定点基因或转录操作的常规手段。尽管如此，CRISPR/Cas9 功能的效率并不能完全预测，而且其变异性的原因也不完全清楚。最近的研究表明，异染色质可能会对 Cas9 的结合和功能产生负面影响。对染色质因子的研究表明，DNA 胞嘧啶-5 甲基化不会直接阻止 Cas9 的结合。然而，核小体在无细胞实验中可以完全阻止 Cas9 进入 DNA，在体内也构成了一个实质性的障碍。除了与开放染色质状态相关外，活性转录还可以通过影响 Cas9 以链特异性方式释放的速率来直接刺激 DNA 切割。有了这些深入的了解和对全基因组染色质和转录状态的更好理解，CRISPR/Cas9 的有效性和可靠性可以得到提高。
