Massively targeted evaluation of therapeutic CRISPR off-targets in cells.
Xiaoguang PanKunli QuHao YuanXi XiangChristian AnthonLiubov PashkovaXue LiangPeng HanGiulia I CorsiFengping XuPing LiuJiayan ZhongYan ZhouTao MaHui JiangJunnian LiuJian WangNiels JessenLars BolundHuanming YangXue LiuGeorge M ChurchJan GorodkinLin LinYonglun LuoPublished in: Nature communications (2022)
Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.
Keyphrases
- genome wide
- single cell
- dna methylation
- rna seq
- induced apoptosis
- high throughput
- cell cycle arrest
- copy number
- randomized controlled trial
- crispr cas
- genome editing
- endothelial cells
- cell death
- endoplasmic reticulum stress
- clinical trial
- dna damage
- study protocol
- signaling pathway
- high glucose
- papillary thyroid
- cell proliferation
- binding protein
- gene therapy
- dna binding
- stress induced