Whole Exome Sequencing of Doxycycline-Inducible Cas9-harboring HEK293. Homo sapiens

Exome sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activitywould be beneficial. To address this need, this manuscript describes a ‘self-inactivating CRISPR (SiC)’system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease ina doxycycline dependent manner. This enables defined, temporal control of Cas9 activity in any cell-typeand also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including: 1. the timed regulation of geneticknockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification ofglycogenes regulating leukocyte-endothelial cell adhesion. 2. Genome-wide lentiviral sgRNA libraryapplications where Cas9 activity is ablated using SiC after allowing pre-determined editing times. Thus,stable knockout cell pools are created for functional screens. 3. Temporal control of Cas9 mediatedediting of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall,SiC enables temporal control of gene editing and may be applied in diverse application including studiesthat aim to reduce off-target genome editing.

针对经一组慢病毒向导RNA处理的细胞进行外显子组测序的结果表明，靶向编辑与脱靶编辑均呈时间依赖性模式。因此，能够实现Cas9活性时间调控的方法具有重要应用价值。为满足这一研究需求，本研究报道了一种「自失活CRISPR（self-inactivating CRISPR，SiC）」系统：该系统由一条单向导RNA构成，可在多西环素依赖的条件下灭活化脓链球菌Cas9核酸酶。该系统可实现任意细胞类型以及体内（in vivo）中Cas9活性的精准时间调控。

实验结果显示，SiC可降低脱靶编辑水平，且对靶向编辑效率的影响相对较小。该工具可适配多种应用场景：
1. 利用慢病毒实现难转染细胞中基因敲除的时序调控，例如在人白细胞中开展相关研究，以鉴定调控白细胞-内皮细胞黏附的糖基因；
2. 全基因组慢病毒单向导RNA（single guide RNA，sgRNA）文库应用：在达到预设编辑时长后，通过SiC终止Cas9活性，从而构建可用于功能筛选的稳定基因敲除细胞池；
3. 体内髓系与淋巴系细胞的Cas9介导编辑时序调控，可应用于小鼠外周血与骨髓组织中的相关编辑过程。

综上，SiC可实现基因编辑的时间调控，有望应用于包括降低脱靶基因组编辑在内的多种研究场景。