Programmable RNA targeting with clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas7-11 in zebrafish embryos and mammalian cells
TL;DR
Findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.
Abstract
The CRISPR/Cas7-11 system is a recently characterized RNA targeting tool that exhibits low toxicity, minimal off-target effects, and reduced collateral RNA cleavage. While Cas7-11 has been recently tested in zebrafish embryos as ribonucleoprotein (RNP) complexes, its use as mRNA–single guide RNA (sgRNA) formulations and its application in human embryonic stem cells (hESCs) have not been explored. In this study, we evaluated the efficiency and specificity of Cas7-11 in zebrafish embryos and hESCs. In zebrafish,injection ofCas7-11and sgRNA resulted in evident reductions of target transcripts, accompanied by clear phenotypic outcomes. Exogenous EGFP mRNA levels were reduced to 31.90%, while endogenous tbxta was reducedto 19.89%, and tyrosinase to 33.18%, respectively, resulting in the no-tail phenotype and reduced pigmentation. Theno-tail phenotype was partially rescued by mRNA overexpression. RNA sequencing confirmed minimal off-target effects. Moreover, Cas7-11 decreased exogenous Gaussia luciferase mRNA in human embryonic and HEK293T cells, to 23.52% and to 63.75%, respectively, and correspondingly reduced luciferase activity, without collateral cleavage activity. Targeting pluripotency factor genes, OCT4 and SOX2, in hESCs decreased their mRNA levels and induced differentiation. Collectively, these findings establish Cas7-11 as a precise and efficient RNA knockdown tool for functional studies in embryonic development and stem cell biology, providing a versatile alternative to DNA-based gene-editing approaches.
