Saturation resistance profiling of EGFR variants against tyrosine kinase inhibitors using prime editing

Abstract

Variants of uncertain significance (VUS) hamper the clinical application of genetic information. For example, in treating lung cancer with tyrosine kinase inhibitors (TKIs), many epidermal growth factor receptor (EGFR) variants remain classified as VUS with respect to TKI sensitivity1,2. Such incomplete resistance profiles hinder clinicians from selecting optimal therapeutic agents3,4. A high-throughput approach that can evaluate the functional effects of single nucleotide variants (SNVs) could reduce the number of VUS. Here we introduce SynPrime, a method based on prime editing that enabled the generation and functional evaluation of 2,476 SNVs in theEGFRgene, including 99% of all possible variants in the canonical tyrosine kinase domain (exons 18 to 21). We determined resistance profiles of 95% (= 1,726/1,817) of all possible EGFR protein variants encoded in the whole tyrosine kinase domain (exons 18 to 24) against afatinib, osimertinib, and osimertinib in the presence of the co-occurring mutation T790M, in PC-9 cells. SynPrime, which uses direct sequencing of endogenous regions to identify SNVs, provided more accurate functional evaluations than a guide RNA abundance-based approach. Our study has the potential to substantially improve the precision of therapeutic choices in clinical settings and contribute to addressing the issue of VUS by being applied to other genes.

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

“…Another effort examined dropout phenotypes from nonsense mutations targeted to one essential gene ( RPL15 ) with the PE2 approach and found that variant effects were categorized as depleting more often when endogenous site sequencing was used to determine phenotypes than without (80% of nonsense variants called as detrimental while only 32% were called using epegRNA abundances; 25 total variants targeted). 30 When evaluating the same gene in our StopPR screen, we observed negative growth phenotypes (Z < −2) for 77.8% (7/9) of targeted nonsense variants with at least one stop epegRNA without endogenous target site sequencing. Our results therefore compare favorably to contemporary platforms and demonstrate the ability to measure high-specificity and reliable phenotypes from epegRNA abundance alone.…”

SynDesign: web-based prime editing guide RNA design and evaluation tool for saturation genome editing

“…Recently, we used SynDesign to develop a saturation resistance profiling library of the EGFR gene against various the chemotherapy drugs, afatinib and osimertinib. A SGE library was designed and evaluated to profile over 2476 SNVs in the EGFR gene that included 95% (1726/1817) of the possible protein variants in the tyrosine kinase domain (exons 18–21) which represents an important functional hotspot for cancer-related mutations ( 4 , 14 ). SynDesign was able to compile a list of all feasible pegRNAs targeting every position in the EGFR gene to systematically evaluate this important oncogene.…”

“…Notably, introducing a synonymous mutation adjacent to the intended edit on the target DNA have been shown to improve on-target prime editing efficiency by diffusing the local cellular MMR activity ( 3 ). Additionally, the synonymous mutation can serve as an indicator of properly edited targets following deep sequencing ( 4 ). Taken together, the application of prime editing for saturation genome editing (SGE) can be a powerful tool in conducting systematic functional screening of variants for target genes of interest, especially for investigating variants of uncertain significance (VUS) that represent a major portion of the single nucleotide variants (SNVs) on the ClinVar and COSMIC databases, 95% and 99%, respectively (Figure 1 ).…”