Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer

Abstract

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations.

Fig. 1. Generation of DNA repair gene knock-outs in human intestinal stem cell cultures.

Targeting strategy for the generation of MLH1 (A) and NTHL1 (B) knockout organoids using CRISPR-Cas9 genome editing. sgRNA, single guide RNA. (C) qRT-PCR for MLH1 in normal and MLH1^KO organoids. Expression was normalized to GAPDH. Mean and SD (error bars) of n = 3 independent experiments are indicated. (D) Same as in (C), but for NTHL1. (E) Western blot analysis of MLH1 expression in normal and MLH1^KO organoids (representative from n = 3). Tubulin was used as a loading control. The asterisk indicates a background band. (F) Same as in (E), but for NTHL1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Fig. 2. Mutational burden in DNA repair–deficient human colonic stem cells.

(A) Number of mutations accumulated in the absence of the indicated DNA repair proteins per day. Base substitutions subdivided by mutation type and INDELs are shown. (B) Size distribution of the observed INDELs per genotype. A negative value indicates deletions and a positive value indicates insertions. (C) Number of INDELs located in simple repeats per genotype. Indicated are the number of repetitive subunits surrounding an inserted or deleted subunit. A value of 0 indicates that the INDEL is not located within a simple repeat.

Fig. 3. Nonrandom genomic distribution of base substitutions in DNA repair–deficient organoids.

(A) Shown for each genotype are enrichment and depletion of base substitutions in the genomic regions that are replicated at the indicated stages during S phase of the cell cycle. Asterisks indicate a significant enrichment or depletion (P < 0.05, one-sided binomial test). (B) Relative levels of each base substitution type in the leading and lagging DNA strands are shown for each genotype. Asterisks indicate a significant difference (P < 0.05, two-sided Poisson test).

Fig. 4. Signatures of mutational processes in DNA repair–deficient organoids.

(A) (Left) Mutational spectra of all base substitutions observed for each genotype. Different mutation types and the direct sequence context are indicated. (Right) Number of mutations per genome per day that can be explained by the indicated mutational signatures for each genotype. (B) Heat map showing the cosine similarity scores for each indicated sample and COSMIC signature. The samples have been clustered according to the similarity score with each signature. The signatures have been ordered according to their similarity, such that very similar signatures cluster together. Arrows indicate signatures that have been associated with deficiency in DNA MMR in pan-cancer analyses (1). MSI, microsatellite instability. (C) Mutations that have been introduced or identified in NTHL1. The blue diamond indicates the site where the selection marker was introduced by gene targeting in the organoids. The red diamond denotes the nonsense germline mutation that was identified in a patient with breast cancer (PD13297a). LOH, loss of heterozygosity.