A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs
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ORIGINAL PAPER
A Novel White‑to‑Blue Colony Formation Assay to Select for Optimized sgRNAs Chaogang Wei1 · Tong Chen1 · Yueyue Zhang1 · Yanfeng Wang1 · Dai Shi1 · Zhen Jiang1 · Kai Li2 · Li Xiao2 · Junkang Shen1 Accepted: 7 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to target genomic DNA sequences as “molecular scissors” with the guidance of sgRNAs. However, the target-specific activities of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently. Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for optimized sgRNAs. Keywords CRISPR/Cas9 · Blue/white · Optimized sgRNA · Gene editing · EphA2
Introduction The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated genome editing system is a microbial immune system that applies RNA-guided nucleases to cleave exogenous DNA. Three types of CRISPR systems have been identified from bacterial and archaeal hosts, and Chaogang Wei and Tong Chen have contributed equally to the work. Li Xiao and Junkang Shen are co-corresponding authors.
the CRISPR-associated (Cas) protein is necessary in this system. The type II CRISPR/Cas system, consisting of a single-guide RNA (sgRNA) and Cas9 protein, has been widely used in biomedical research [1–3]. SgRNA, which is fused together by CRISPR RNA (crRNA) and transactivating crRNA (tra-crRNA), can direct the Cas9 endonuclease to cleave specific target DNA sites [4]. The Cas9 protein can be guided to modify and cleave target DNA sequences by mainly relying upon sgRNA sequences with base-pair complementarity to the DNA sequences, and the target specificity is mainly determined by sgRNA sequences [5].
* Li Xiao [email protected]
Dai Shi [email protected]
* Junkang Shen [email protected]
Zhen Jiang [email protected]
Chaogang Wei [email protected]
Kai Li [email protected]
Tong Chen [email protected]
1
Department of Radiology, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou 215004, Jiangsu, People’s Republic of China
2
Center of Laboratory, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou 215004, Jiangsu, People’s Republic of China
Yueyue Zhang [email protected] Yanfeng Wang [email protected]
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