Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, Christiana Care Health System, Newark, DE 19713, USA.
Department of Medical and Molecular Sciences, University of Delaware, Newark, DE 19716, USA.
Genes (Basel). 2020 Sep 30;11(10):1160. doi: 10.3390/genes11101160.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.
簇状规律间隔短回文重复（CRISPR）/Cas 基因编辑系统使分子遗传学家能够更有效地、更精确地操作原核生物和真核生物的基因组。CRISPR/Cas 通过降解入侵的病毒基因组，为细菌细胞提供适应性免疫。通过将这种活性民主化到人类细胞中，可以敲除特定基因以使其功能失效并修复错误。后一种活动需要单链供体 DNA 模板的参与，该模板提供遗传信息，以执行同源定向修复（HDR）过程中的校正。在这里，我们利用已建立的无细胞提取物系统来确定供体 DNA 模板长度对 CRISPR 指导的基因编辑产物多样性的影响。该模型系统使我们能够观察到该反应的所有结果，并揭示出供体模板长度会影响反应的效率以及伴随的易错产物的类别。对产物的仔细测量揭示了一类包含校正模板以及插入和缺失（indels）的易错事件。我们的数据为那些旨在将 CRISPR/Cas 从实验室转化为临床的人提供了基础信息。
