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.
Unlike traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here, we show in a mouse model of tyrosinemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single guide RNA can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes in the liver, and rescued weight loss in the animals. We also generated Fah+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABE. Our findings suggest that adenosine base editing can be used for the correction of genetic disease in adult animals.
Defining the genetic drivers of cancer progression is a key in understanding disease biology and developing effective targeted therapies. Chromosome rearrangements are a common feature of human malignancies, but whether they represent bona fide cancer drivers and therapeutically actionable targets, requires functional testing. Here, we describe the generation of transgenic, inducible CRISPR-based mouse systems to engineer and study recurrent colon cancer-associated EIF3E–RSPO2 and PTPRK–RSPO3 chromosome rearrangements in vivo. We show that both Rspo2 and Rspo3 fusion events are sufficient to initiate hyperplasia and tumour development in vivo, without additional cooperating genetic events. Rspo-fusion tumours are entirely Wnt-dependent, as treatment with an inhibitor of Wnt secretion, LGK974, drives rapid tumour clearance from the intestinal mucosa without effects on normal intestinal crypts. Altogether, our study provides direct evidence that endogenous Rspo2 and Rspo3 chromosome rearrangements can initiate and maintain tumour development, and indicate a viable therapeutic window for LGK974 treatment of RSPO-fusion cancers.
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.
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