CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.

The majority of colorectal cancers (CRCs) show hyperactivated WNT signaling due to inactivating mutations in the APC tumor suppressor. Genetically restoring Apc suppresses WNT and induces rapid and sustained tumor regression, implying that re-engaging this endogenous tumor suppressive mechanism may be an effective therapeutic strategy. Here, using new animal models, human cell lines, and ex vivo organoid cultures, we show that Tankyrase (TNKS) inhibition can control WNT hyperactivation and provide long-term tumor control in vivo, but that effective responses are critically dependent on how APC is disrupted. Mutant APC proteins truncated within the Mutation Cluster Region (MCR) region physically engage the destruction complex and suppress the WNT transcriptional program, while early APC truncations (i.e. Apc Min) show limited interaction with AXIN1 and β-catenin, and do not respond to TNKS blockade. Together, this work shows that TNKS inhibition, like APC restoration, can reestablish endogenous control of WNT/β-catenin signaling, but that APC genotype is a crucial determinant of this response.

The WNT pathway is a fundamental regulator of intestinal homeostasis, and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer. To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that although WNT suppression blocks tumor growth in most organoid and in vivo colorectal cancer models, the accumulation of colorectal cancer–associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of TP53 and SMAD4, transient TGFβ exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent colorectal cancer growth, and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs. Significance: Colorectal and intestinal cancers are driven by mutations in the WNT pathway, and drugs aimed at suppressing WNT signaling are in active clinical development. Our study identifies a mechanism of acquired resistance to WNT inhibition and highlights a potential strategy to target those drug-resistant cells. This article is highlighted in the In This Issue feature, p. 1426

KRAS is the most frequently mutated oncogene in cancer.Tumor sequencing has revealed a complex spectrum of KRAS mutations across different cancer types, yet there is little understanding how specific KRAS alterations impact tumor in initiation, progression, or therapy response. Using highfidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRAS G12C ), pancreas (KRAS G12R ) and colon (KRAS G13D ) cancers. Induction of each mutation in the developing mouse pancreas reveal striking quantitative and qualitative differences in the degree of ductal transformation and pre-malignant progression. Further, using organoid models we show that KRAS G13D mutants respond to EGFR inhibition, while the anti-proliferative effect of KRAS G12C -selective inhibitors can be overcome by upstream EGFR signaling. Together, these new mouse strains provide an ideal for investigating KRAS biology in vivo, and for developing pre-clinical precision oncology models of KRAS-mutant pancreas (G12R), colon (G13D), and lung (G12C) cancers.KRAS mutations in tumor development. However, these models alone do not recapitulate the spectrum of KRAS alterations in human cancer. Here we describe an efficient pipeline for engineering allelic series of conditional alleles that significantly expands repertoire of pre-clinical KRASdriven cancer models. Using high-fidelity CRISPR targeting in embryonic stem cell (ESC)-based mouse models (GEMM-ESCs) 11-14 , we engineered six new LSL-Kras mutant alleles (G12V, G12C, G13D, G12R, G12A, G12S) that represent the most frequent mutations at the G12/G13 hotspot, after G12D. Guided by clinical data, we generated conditional mice representing three tissue-selective alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C) and show that, even subtle mutational changes in the Kras oncogene, have a profound impact on tumor initiation in the pancreas.Conditional animal models, such as Lox-Stop-Lox (LSL)-Kras G12D and LSL-Kras G12Vgeo mice developed almost 20 years ago 9,10 , have been critical tools to dissect the role of . CC-BY-NC-ND 4.

Base editing (BE) can be applied to characterize single nucleotide variants (SNVs) of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular BE-activity ‘sensors’ that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor–sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated SNVs in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models, and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput BE screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.