Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes

Logan O Gauthier; Ziyi Wang; Kenneth K Ng; Shushu Huang; Yafei Mao; Monkol Lek; Kaiyue Ma

doi:10.1016/j.xpro.2025.103909

Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes

STAR Protoc. 2025 Jun 19;6(3):103909. doi: 10.1016/j.xpro.2025.103909. Online ahead of print.

Authors

Logan O Gauthier¹, Ziyi Wang², Kenneth K Ng¹, Shushu Huang¹, Yafei Mao², Monkol Lek³, Kaiyue Ma⁴

Affiliations

¹ Department of Genetics, Yale School of Medicine, New Haven, CT 06510, USA.
² Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200230, China.
³ Department of Genetics, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address: monkol.lek@yale.edu.
⁴ Department of Genetics, Yale School of Medicine, New Haven, CT 06510, USA; Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200230, China. Electronic address: kaiyue.ma@aya.yale.edu.

Abstract

Determining the functional impacts of disease-causing genetic variants presents consistent challenges in the genetic disease field. Here, we present a protocol for implementing saturation mutagenesis-reinforced functional assays to generate functional scores for small-sized variants in disease-related genes. We describe procedures for nucleofection to establish cell line platforms, programmed allelic series with common procedures (PALS-C) cloning for saturation mutagenesis, fluorescence-based cell sorting, next-generation sequencing, and functional score generation. This framework holds potential for high-throughput and cost-effective interpretation of unresolved variants in a broad array of disease genes. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.

Keywords: CRISPR; Cell Biology; Flow Cytometry; Genetics; Genomics; High-Throughput Screening; Molecular Biology; Sequencing.