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Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

ABSTRACT: CRISPR-Cas, the immune system of bacteria and archaea, has been widely harnessed for genome editing, including gene knockouts and knockins, single-base editing, gene activation, and silencing. However, the molecular mechanisms underlying fluctuations in the genome editing efficiency of crispr in various cells under different conditions remain poorly understood. In this work, we found that Cas9 can be ac(et)ylated by acetyl-phosphate or acyl-CoA metabolites both in vitro and in vivo. Several modifications are associated with the DNA or sgRNA binding sites. Notably, ac(et)ylation of Cas9 driven by these metabolites in host cells potently inhibited its binding and cleavage activity with the target DNA, thereby decreasing Crispr genome editing efficiency. This study provides more insights into understanding the effect of the intracellular environment on genome editing application of crispr with varying efficiency in hosts.

SUBMITTER: Zhao L

PROVIDER: S-EPMC9802096 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

Zhao Li L You Di D Wang Ting T Zou Zhen-Ping ZP Yin Bin-Cheng BC Zhou Ying Y Ye Bang-Ce BC

PNAS nexus 20221101 5

CRISPR-Cas, the immune system of bacteria and archaea, has been widely harnessed for genome editing, including gene knockouts and knockins, single-base editing, gene activation, and silencing. However, the molecular mechanisms underlying fluctuations in the genome editing efficiency of crispr in various cells under different conditions remain poorly understood. In this work, we found that Cas9 can be ac(et)ylated by acetyl-phosphate or acyl-CoA metabolites both <i>in vitro</i> and <i>in vivo</i> ...[more]

PMID: 36712324

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Project description:BackgroundProgression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.MethodsIL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.ResultsWe demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.ConclusionsIL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.

Dataset Information

Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

Publications

Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing.

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