In vivo functional screening for systems-level integrative cancer genomics

Abstract

With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.

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Fig. 1: Transposon tools for genome-wide screening in mice.
Fig. 2: Principles and design evolution of RNAi-mediated gene knockdown.
Fig. 3: CRISPR tools and applications.
Fig. 4: Principles of in vivo CRISPR screening.
Fig. 5: Functionalizing cancer evolution through in vivo genetic interaction screening.

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Acknowledgements

The authors apologize to colleagues whose work was not acknowledged owing to space limitations. C.J.B. is funded by the Max-Eder Program of Deutsche Krebshilfe (70113377) and The Care for Rare Foundation. D.S. is supported by the German Research Foundation (SA1374/4-2; SFB 1321) and the European Research Council (Consolidator Grant 648521). R.R. receives funds from the Deutsche Krebshilfe (70112480), the German Research Foundation (RA 1629/2-1; SFB1243; SFB1321; SFB1335) and the European Research Council (Consolidator Grant 819642 PACA-MET and MSCA-ITN-ETN 861196).

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J.W., C.J.B., D.S. and R.R. all researched data for the article, provided a substantial contribution to discussions of the content and contributed to writing the article and to the review and/or editing of the manuscript before submission.

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Correspondence to Roland Rad.

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Nature Reviews Cancer thanks L. Dow, J. Jonkers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Epitranscriptional

The post-transcriptional processing of RNA molecules, such as methylation of certain bases, which can influence RNA localization, stability, translation and decay.

Nucleotide transversions

DNA point mutations that lead to purine to pyrimidine substitutions, or vice versa (A ↔ C, A ↔ T, C ↔ G, G ↔ T).

Nucleotide transitions

DNA point mutations that lead to the exchange of two purines (A ↔ G) or two pyrimidines (C ↔ T).

Saturation mutagenesis

The introduction of a large number of mutations within a specific genomic region or even a whole genome to identify all functional elements producing a specific phenotype.

Slow transforming retroviruses

An oncogenic retrovirus that induces tumorigenesis through insertional mutagenesis. In contrast to acute transforming retroviruses, its genome does not contain viral oncogenes.

Promoters

DNA sequences that, upon binding of transcription factor proteins, initiate transcription of downstream genes by an RNA polymerase.

Enhancers

Cis-acting regulatory DNA elements that increase the activity of promoters and, thus, the transcription rate of genes (or other transcribed elements) in a cell type-specific but position-independent and orientation-independent manner.

Hypomorphic

A mutation that causes a partial loss of gene function, such as reduced expression or lower substrate affinity.

Gene-trapping elements

Regulatory cassettes consisting of splice acceptor sites and polyadenylation signals capable of terminating transcription prematurely from intronic positions.

Local hopping

The preference of DNA transposon systems to reintegrate close to their excision site.

Missense mutations

DNA point mutations leading to amino acid substitution.

Silent mutations

DNA point mutations that do not lead to an amino acid change owing to the redundancy of the genetic code.

Haploinsufficiency

The cause of dominant deleterious gene action in diploid organisms. For haploinsufficient tumour suppressors, monoallelic inactivation is sufficient to support oncogenesis. By contrast, for ‘classic’ tumour suppressors, inactivation of both alleles is required for tumorigenesis (recessive phenotype).

Loss of heterozygosity

(LOH). The loss of the second (wild-type) allele for monoallelic mutations. In somatic cells, LOH can result from genetic deletions, mitotic recombination between non-sister chromatids or mitotic non-disjunction of chromosomes.

Mitotic recombination

The DNA exchange between sister or non-sister homologous chromatids during mitosis.

Cooperation

A process in which two or more entities (for example, cancer genes) act together for their mutual benefit (for example, promoting tumorigenesis).

Insulator

A cis-acting regulatory DNA element preventing activation of gene promoters by distal enhancers (enhancer-blocking insulators) and/or euchromatin silencing through spread of neighbouring heterochromatin (barrier insulators).

Hydrodynamic tail vein injection

(HTVI). A method for in vivo delivery of small compounds, such as DNA or nanoparticles, into hepatocytes by rapid injection of a large volume of liquid into the mouse tail vein, which induces cardiac congestion and a retrograde liquid flux into the liver.

Non-homologous end joining

(NHEJ). An error-prone repair mechanism for DNA double-strand breaks.

Homology-directed repair

(HDR). An error-free repair mechanism of DNA double-strand breaks, which requires an intact DNA template strand with significant homology to the region surrounding the to-be-repaired double-strand break.

Cas9 nickase

(Cas9n). A Cas9 variant with only one functional endonuclease domain that is capable of inducing DNA single-strand but not double-strand breaks.

Nonsense mutations

DNA point mutations resulting in a stop codon and subsequent premature termination of translation.

Splice site mutations

DNA point mutations within splice sites that can disrupt RNA splicing, resulting in loss of exons (‘exon skipping’) or inclusion of introns (‘intron retention’).

Dead Cas9

(dCas9). A catalytically inactive Cas9 variant that can bind but not cleave DNA owing to engineered deleterious mutations in both endonuclease domains.

Synthetic lethality

An extreme form of negative genetic interaction, in which the loss of two genes leads to cell death, whereas the loss of each gene alone has milder effects or no effect on cellular fitness.

Unique molecular identifiers

(UMIs). High-dimensional molecular barcodes for detection, quantification and tracking of RNA or DNA input molecules. Incorporation of UMIs into single guide RNA (sgRNA) libraries enables measurement of the behavioural consistency of identical sgRNAs within a screen.

Cellular fitness

The ability of cancer cells to flourish within their niche and compete with neighbouring clones. The fitness of a given genotype can be different in different selective environments.

Clonal sweep

A reduction in diversity through a beneficial variation (for example, mutation in a tumour subclone) that eliminates other variants owing to strong positive selection.

Evolutionary constraints

Limitations or biases on the evolution of specific adaptative phenotypes, forcing, for example, cancer cells to evolve along specific molecular paths or sequences.

Parallel evolution

The independent evolution of similar traits in lineages originating from a common ancestor, for example, acquisition of alterations in the same genes or pathways in subclones evolving from a common cell of origin within a cancer.

Convergent evolution

The independent development of similar traits in separate lineages without a common ancestor, for example, acquisition of genetic or epigenetic alterations in the same genes or pathways in tumours from different patients.

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Weber, J., Braun, C.J., Saur, D. et al. In vivo functional screening for systems-level integrative cancer genomics. Nat Rev Cancer (2020). https://doi.org/10.1038/s41568-020-0275-9

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