SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution
Yao Yao1, Zhiwei Zhou1, Xiaoling Wang1
1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; Tianjin Institutes of Health Science, Tianjin 301600, China.
Cell Genomics
|June 18, 2024
Summary
This study introduces SpRY mutagenesis and base editing screens for high-resolution identification of functional regulatory elements and genetic variants. This advanced CRISPR screening method precisely maps causal variants, improving gene expression studies.
Area of Science:
- Genomics
- Molecular Biology
- Genetic Engineering
Background:
- CRISPR mutagenesis screens using SpCas9 have limitations in resolution due to Protospacer Adjacent Motif (PAM) sequence requirements.
- Identifying functional cis-regulatory elements and genetic variants impacting gene expression is crucial for understanding biological processes.
Purpose of the Study:
- To develop and validate a high-resolution CRISPR screening method using the near-PAMless SpRY variant for identifying functional regulatory elements and genetic variants.
- To apply this method to a specific genome-wide association study (GWAS) locus associated with red blood cell traits.
Main Methods:
- Utilized saturated SpRY mutagenesis and base editing screens for high-resolution functional genomics.
- Applied the SpRY screening methodology to a GWAS locus (10q22.1) linked to red blood cell traits.
- Integrated Bayesian statistical fine-mapping for variant analysis.
Main Results:
- Demonstrated that saturated SpRY screens can accurately identify functional regulatory elements and essential genetic variants at single-base resolution.
- Identified potential enhancers regulating HK1 gene expression within the 10q22.1 GWAS locus, including those lacking typical chromatin signatures.
- Pinpointed multiple causal variants within the locus missed by traditional fine-mapping methods.
Conclusions:
- Saturated SpRY mutagenesis and base editing screens offer a powerful, high-resolution approach for interrogating non-coding genomic elements.
- This methodology significantly enhances the ability to identify functional variants and regulatory elements, complementing existing CRISPR screening techniques.
