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Direct-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen.

Genome biology (2020-06-10)
Qingkai Song, Ke Ni, Min Liu, Yini Li, Lixia Wang, Yingying Wang, Yingzheng Liu, Zhenxing Yu, Yinyao Qi, Zhike Lu, Lijia Ma
ABSTRACT

CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription, and epigenetic modifications in genetic screens. However, it remains challenging to assay the complex molecular readouts after perturbation at high resolution and at scale. By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow.

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RPMI-1640 Medium, With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture