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CRISPR/Cas9 technology is a powerful tool for gene editing in mammalian cells. However, the efficiency of gene editing in certain types of cells, especially suspension cells such as several blood-lineage cell lines, is extremely low. This is due to a variety of factors including low transfection efficiency of CRISPR/Cas9 reagents, cytotoxicity, and low or undesired gRNA and Cas9 activities in the cell lines. By optimizing the protocol and transfection conditions, we were able to successfully generate a spectrum of gene modifications in blood-lineage cells. Here, we describe techniques optimized for genome editing in some difficult-to-transfect blood-lineage cell lines and the types of modifications achieved: (1) For leukemia cell lines such as KG-1 and MOLM-13 cells, we compared DNA-based versus RNP-based Cas9 delivery efficiency, and found that plasmid-based Cas9 transfection resulted in cytotoxicity. In contrast, the RNP-based route demonstrated significantly lower cytotoxicity and higher efficiency of gene editing. Furthermore, we found that the application of a DNA-dependent protein kinase inhibitor dramatically enhanced the homology directed repair (HDR) in these cell lines; (2) For T lymphocyte cell lines such as Jurkat cells, we could efficiently delete a targeted 5kb regulatory region of a critical gene. We were also able to sequentially knockout (KO) two genes (double KO) in Jurkat cells using a Cas9-plasmid protocol; (3) In the case of the lymphoblastic cell line T2, we successfully created a large fragment knock-in, as well as a gene knockout cell line model, through DNA-based CRISPR/Cas9 delivery. Our studies demonstrate that for CRISPR/Cas9 genome editing in blood-lineage cells, different cell lines may require different approaches or modified protocols to deliver the CRISPR/Cas9 components for efficient and successful modifications of the targeted genes. Citation Format: Huanyu Jin, Zhongsheng Yu, Vladimir Pak, Yin Zhang, Padmaja Tummala, Pavithra Rajeswaran, Diana Nguyen, Monika Maleszewska, Jinling Li, Charles Cao, Ruby Yanru Chen-Tsai. Full spectrum of in vitro disease models with optimized CRISPR-Cas9 gene editing in difficult-to-transfect blood-lineage cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3117.

Abstract 3117: Full spectrum of in vitro disease models with optimized CRISPR-Cas9 gene editing in difficult-to-transfect blood-lineage cells