CRISPR screening

CRISPR Screening: A Look Into the NOTCH Pathway

CRISPR screening

CRISPR screening is a useful experimental approach used to facilitate the discovery of genes or genetic sequencing. According to a study done by researchers in the University of Toronto, long-tail mutations or less frequent mutations play a role in human head and neck squamous cell carcinoma (HNSCC) cases. They were able to discover this by looking closely into available somatic mutation data for HNSCC and doing a follow up on candidate long-tail HNSCC-related alterations with CRISPR-Cas9-based gene editing experiments in mouse models. This confirms the power of integrating cancer genomics with mouse modeling in vivo CRISPR screening to uncover tumor-suppressive pathways in the long tail of cancer-associated mutations.

Starting from previously generated somatic mutation data for HNSCC, they focused in on 484 genes having recurrent mutations in HNSCC that are less common than those affecting driver genes such as TP53. With the data gathered, they were able to narrow in on a small set of apparent tumor suppressor genes. This collection includes at least two genes that feed into the NOTCH pathway, which plays an important role in cell proliferation, differentiation, and survival.

Oncogenic Mutations Converge Onto NOTCH

According to their senior and corresponding author Daniel Schramek who is also a researcher affiliated with Mount Sinai Hospital in Toronto and the University of Toronto, their experiments showed that oncogenic mutations in 67 percent of human HNSCC cases converge onto the NOTCH signaling pathway. This makes NOTCH inactivation a hallmark of HNSCC. 

Schramek also said that “the most common genetic alterations in HNSCC affect p53 (71 percent), FAT1 (23 percent), CDKN2A (22 percent), PIK3CA (18 percent), NOTCH1 (17 percent), and HRAS (6 percent), followed by a ‘long tail’ of hundreds of individually rare mutations, most of which lack biological or clinical validation.”

CRISPR Screening

Based on the data on hand, they developed lentiviral single-guide RNA (sgRNA) libraries for in vivo CRISPR-based knockout screens in mice. They did this by searching for alterations that pushed HNSCC development in mice forward compared to control mice screened with more than 400 non-gene-targeting sgRNAs and another 414 sgRNAs targeting random genes not guided by the HNSCC data.

The researchers then validated the mouse screening strategy and injected sgRNA libraries into mouse embryos that carried conditional HNSCC-related mutations in PIK3CA, HRAS, or p53 or mice expressing a high-risk version of human papillomavirus. They found that mice missing genes from the long tail mutation set developed HSCC tumors as well as cutaneous squamous carcinoma. On the other hand, they found that mice undergoing CRISPR screening with control libraries did not develop the cancers over the course of a year. 

Subsequent experiments suggested that HNSCC was not spurred on in mice carrying conditional PIK3CA mutations over four months when breast cancer-related genes were altered by gene editing.

They also noted that NOTCH2 and NOTCH3 genes turned up in a group of 15 potential tumor suppressor genes, each altered by at least two of the sgRNAs in the mouse tumors. They also found that ADAM10 and AJUBA subsequently interfered with NOTCH signaling when mutated.

Taking the findings back into the human HNSCC context, they saw mutations in AJUBA in 7.5 percent of HNSCC cases assessed for the Cancer Genome Atlas project. They also noted that heterozygous mutations were even more common in HNSCC coming in at more than 11 percent of tumors for the AJUBA gene, and at 12.7 percent for ADAM10.

The authors thereby look at NOTCH as a dysregulated pathway in HNSCC based on their research.

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