Rapid generation of precision preclinical cancer models using regulatable in vivo base editing

Abstract

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

Applied models and molecular characteristics of small cell lung cancer

“…Since the 2010s, CRISPR/Cas9 GEMMs have been applied in cancer research [86,87], and significant breakthroughs occurred in 2023. These innovations include the development of mice capable of CRISPR/Cas9 base editing [88]…”

Applications of CRISPR-Cas9 for advancing precision medicine in oncology: from target discovery to disease modeling

“…Another exciting new development in precision disease modeling is the introduction of base and prime editors that can directly introduce disease-specific gene mutation without the need for the DNA template ( Komor et al, 2016 ; Gaudelli et al, 2017 ; Rees and Liu, 2018 ; Chen and Liu, 2023 ). These models serve as valuable tools in identifying and validating patient-specific cancer drivers and resistance mechanisms ( Smurnyy et al, 2014 ; Yin et al, 2019 ; Katti et al, 2022b ).…”

“…Its high efficiency in introducing precise point mutation, without introducing DSB or requiring a donor template, makes it an ideal tool for cancer drug discovery, as most of the cancer variants are point mutations. Within a relatively short time since its development, base editing has been successfully implemented in the generation of cellular, organoid, and animal models ( Komor et al, 2017 ; Musunuru et al, 2021 ; Rothgangl et al, 2021 ; Li et al, 2022a ; Katti et al, 2022b ). For instance, Li et al (2020b) used an adenine base editor to successfully correct oncogenic point mutations on the TERT promoter in glioblastoma cells, leading to tumor growth inhibition.…”

“…In addition to in vitro models, the base editor was quickly implemented in generating in vivo models. For example, Katti et al (2022b) developed a doxycycline-inducible base editing system to generate rapid in vivo models carrying disease-specific mutations in various organs, such as the lung, intestine, and pancreas. Moreover, base editing could achieve high mutational frequency in animal models compared to CRISPR nuclease ( Rees and Liu, 2018 ), underscoring the potential of this tool in elucidating the function of disease-causing mutants in driving pathogenesis.…”