On 16 December 2019, the research paper entitled "PASTMUS: mapping functional elements at single amino acid resolution in human cells" from Prof. Wei Wensheng's group was published online in Genome Biology

It is of great importance for the identification of functional elements for a protein of interest to achieve a mechanistic understanding. Traditional methods mainly rely on in vitro biochemical assays, such as coimmunoprecipitation (Co-IP) combined with truncation mutagenesis; however, these techniques have a low resolution, and none of them is performed in native biological contexts. Previous studies include the screening of cells expressing cDNAs containing various missense mutations, screening through generating point mutations, screening of tiling library followed by NGS (next-generation sequencing) on enriched sgRNAs, and a recent approach named “tag-mutate-enrich”. Most of these methods require the exogenous expression of cDNAs. They are also limited by the coverage of the actual amino acids of the target, the types of mutation, or the resolution of the functional map. After all, most of these methods are not designed to study mutations that are genetically recessive. There is no existing method that could potentially assess all amino acids of a given protein for their functional importance, especially in the native biological contexts.

Here, we report a strategy, PArsing fragmented DNA Sequences from CRISPR Tiling MUtagenesis Screening (PASTMUS), which provides a streamlined workflow and a bioinformatics pipeline to identify critical amino acids of proteins in their native biological contexts. Using this approach, we map six proteins—three bacterial toxin receptors and three cancer drug targets and acquire their corresponding functional maps at amino acid resolution. Moreover, PASTMUS strategy is also suited for acquiring functional maps of regulatory elements, such as non-coding RNA, promoters, and enhancers.

Xinyi Zhang, Di Yue, Dr. Ying Liu, Dr. Yinan Wang, and Dr. Yuexin Zhou are the co-first authors of the paper. Prof. Wei Wensheng is the corresponding author. This project was supported by funds from the National Science Foundation of China, the Beijing Municipal Science & Technology Commission, the Beijing Advanced Innovation Center for Genomics at Peking University, the Peking-Tsinghua Center for Life Sciences, the National Science Foundation of China, and the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China.

PASTMUS workflow and bioinformatics pipeline to identify critical residues of proteins