Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells.
Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells.
复制标题
DOI：
10.1016/j.xpro.2022.101675
复制
发表时间：
2022-12-16
期刊：
影响因子：
--
通讯作者：
Bielinsky, Anja-Katrin
中科院分区：
文献类型：
--
作者：
Lin, Kevin;Chang, Ya-Chu;de Velasco, Ezequiel Marron Fernandez;Wickman, Kevin;Myers, Chad L.;Bielinsky, Anja-Katrin
合并的慢病毒CRISPR-Cas9筛选用于评估许多单基因敲除对化合物的差异敏感性或抗性。在这里，我们提出了一种可扩展的方法，通过利用一个小的定制库进行高通量的化合物筛选。我们描述了在非转化的hTERT RPE-1 TP 53 −/−细胞中进行原理验证化学筛选的步骤，与全基因组筛选相比，具有更高的覆盖率和更高的时间点分辨率。这种方法可以适用于各种细胞系、化合物和其他聚焦的sgRNA文库。在非转化的人细胞系中进行可扩展的CRISPR-Cas9化学筛选的方案sgRNA文库的慢病毒包装和筛选的化合物剂量优化在1000×覆盖率的中间时间点进行可扩展的化学筛选使用质量控制图进行sgRNA富集或缺失的生物信息学分析进行任何实验方案都需要遵守当地实验室安全和伦理的机构指南。合并的慢病毒CRISPR-Cas9筛选用于评估许多单基因敲除对化合物的差异敏感性或抗性。在这里，我们提出了一种可扩展的方法，通过利用一个小的定制库进行高通量的化合物筛选。我们描述了在非转化的hTERT RPE-1 TP 53 −/−细胞中进行原理验证化学筛选的步骤，与全基因组筛选相比，具有更高的覆盖率和更高的时间点分辨率。这种方法可以适用于各种细胞系、化合物和其他聚焦的sgRNA文库。
Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries. Protocol for scalable CRISPR-Cas9 chemical screens in a non-transformed human cell line Lentiviral packaging of sgRNA library and compound dosage optimization for screens Conducting scalable chemical screens with intermediate timepoints at 1000× coverage Bioinformatics analysis of sgRNA enrichment or dropout with quality control plots Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53−/− cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.
登录
查看更多内容
影响因子：
16.6
作者：
Baxley RM;Leung W;Schmit MM;Matson JP;Yin L;Oram MK;Wang L;Taylor J;Hedberg J;Rogers CB;Harvey AJ;Basu D;Taylor JC;Pagnamenta AT;Dreau H;Craft J;Ormondroyd E;Watkins H;Hendrickson EA;Mace EM;Orange JS;Aihara H;Stewart GS;Blair E;Cook JG;Bielinsky AK
通讯作者：
Bielinsky AK
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
影响因子：
--
作者：
Chen, Shih-Heng;Haam, Juhee;Martin, Negin P
通讯作者：
Martin, Negin P
影响因子：
64.5
作者：
Hart, Traver;Chandrashekhar, Megha;Moffat, Jason
通讯作者：
Moffat, Jason
影响因子：
82.9
作者：
Haapaniemi, Emma;Botla, Sandeep;Taipale, Jussi
通讯作者：
Taipale, Jussi