(1)CRISPR screening在准备阶段需要使用电转的方式扩增文库,并需要NGS测序来确定文库丰度,尽管目前已经有一些library可以选用化学感受态细胞进行扩增,但选用library之前一定要查阅清楚; (2)CRISPR screening实验需要大量的细胞,因此对于一些无法扩增、数量有限且非常珍贵的细胞时,如原代细胞等,CRISPR screening则可能不适用; ...
在设计CRISPR screening实验之前需要思考以下问题： （1）CRISPR screening在准备阶段需要使用电转的方式扩增文库，并需要NGS测序来确定文库丰度，尽管目前已经有一些library可以选用化学感受态细胞进行扩增，但选用library之前一定要查阅清楚； （2）CRISPR screening实验需要大量的细胞，因此对于一些无法扩增、...
(1)CRISPR screening在准备阶段需要使用电转的方式扩增文库,并需要NGS测序来确定文库丰度,尽管目前已经有一些library可以选用化学感受态细胞进行扩增,但选用library之前一定要查阅清楚; (2)CRISPR screening实验需要大量的细胞,因此对于一些无法扩增、数量有限且非常珍贵的细胞时,如原代细胞等,CRISPR screening则可能不适用; ...
High Throughput ScreeningStem CellsPAX6is a key determinant of human neuroectoderm cell fate. Here, we describe a protocol for genome-scale CRISPR screening for use in genetically engineered human pluripotent stem cells (hPSCs). Using the germ layer reporterPAX6and an inducible CRISPR/Cas9 knockout...
Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogat
Fig. 1: CRISPRi screening of prostate cancer risk CREs. aSchematic of rCRE selection and sgRNA design.bDistribution of sgRNAs targeting rCREs or control regions. The barplot in the inset indicates the number of regions in the library.cThe cumulative distribution of depletion p-values of sgRNA...
[11,12]. This screening strategy provides potential targets for synergy, while attenuates the applicable value for monotherapy to a certain extent. To our knowledge, we first identified cyclin-dependent kinase 12 (CDK12) as conservatively required for PCa cells under both normal and AR antagonism ...
Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide ...
Single-cell CRISPR screening is a powerful method for biological discovery. Such screens genetically perturb many genes in parallel and use single-cell RNA sequencing to assess the transcriptomic effects. Now, an in vivo protocol facilitates screens in mouse brain, using adeno-associated virus with ...
This system enables a one-step, high-efficiency protocol for easy lentiviral production and transfection. Following screening, next-generation sequencing (NGS) analysis can be performed to identify candidate genes of interest using the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit, which...