Programmable RNA targeting with clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas7-11 in zebrafish embryos and mammalian cells

Abstract

<title>Abstract</title> <bold>Background</bold> The CRISPR/Cas7-11 system is a recently characterized RNA editing tool that exhibits low toxicity, minimal off-target effects, and reduced collateral RNA cleavage compared with existing methods. Although zebrafish and human embryonic stem cells (hESCs) serve as important models for developmental and regenerative research, the application of Cas7-11 in these model systems has not been systematically explored. <bold>Results</bold> In this study, we evaluated the efficiency and specificity of Cas7-11 in zebrafish embryos and hESCs. In zebrafish, injection of Cas7-11 and sgRNA resulted in evident reductions of target transcripts, accompanied by clear phenotypic outcomes. Exogenous <italic>EGFP</italic> mRNA levels were reduced to 31.90%, while endogenous <italic>tbxta</italic> were reduced to 19.89%, and <italic>tyrosinase</italic> to 33.18%, respectively, resulting in the no-tail phenotype and reduced pigmentation. The no-tail phenotype was partially rescued by mRNA overexpression. RNA sequencing confirmed minimal off-target effects. Moreover, Cas7-11 decreased exogenous <italic>Gaussia luciferase</italic> 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, <italic>OCT4</italic> and <italic>SOX2,</italic> in hESCs decreased their mRNA levels and induced differentiation. <bold>Conclusions</bold> 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.