SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution

• Published in: Cell genomics 2024-07, Vol.4 (7), p.100583, Article 100583
• Main Authors: Yao, Yao, Zhou, Zhiwei, Wang, Xiaoling, Liu, Zhirui, Zhai, Yixin, Chi, Xiaolin, Du, Jingyi, Luo, Liheng, Zhao, Zhigang, Wang, Xiaoyue, Xue, Chaoyou, Rao, Shuquan
• Format: Article
• Language: English
• Subjects: CRISPR-Cas Systems - genetics; Enhancer Elements, Genetic - genetics; Gene Editing - methods; Genome-Wide Association Study - methods; Humans; Mutagenesis; Technology
• ISSN: 2666-979X; 2666-979X
• DOI: 10.1016/j.xgen.2024.100583

CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens. [Display omitted] •Trimming the spacer length from 20 to 19 nt enhances SpRY editing efficiency•SpRY mutagenesis screens faithfully identify functional regulatory sequences•SpRY base editing screens pinpoint causal variants overlooked by statistical fine-mapping•SpRY mutagenesis and base editing screens could complement each other One major caveat when using CRISPR screening to investigate non-coding sequences is low resolution due to limited availability of the PAM sequences. Yao et al. have demonstrated that saturated mutagenesis and base editing screens using the near-PAMless SpRY variant can faithfully identify functional regulatory elements and genetic variants at single-base resolution. This approach complements other CRISPR screen technologies in dissecting non-coding sequences.