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bioRxiv · 10.1101/2022.03.17.484717

Rational design of Cas9 ribonucleoprotein with a "gRNA-shRNA" for multidimensional genome manipulation and enhanced homology-directed repair

qiao, j.·sun, w.·yin, w.·ma, l.·liu, y.
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Abstract

Gene perturbation approaches have evolved as powerful tools for understanding the function of genes and curing inherited diseases. Here, we develop a method that combines the merits of RNAi and CRISPR technology by rational design of Cas9 ribonucleoprotein (RNP) with a "gRNA-shRNA" component. The RNP, termed Cas9-RNAi, has a gRNA containing a 3 extension that can be processed to a functional siRNA via dorsha/dicer enzyme mediated cleavage within cells. We prepared the Cas9-RNAi RNPs by streamline co-expression of Cas9 enzymes and the "gRNA-shRNA" ribonucleotides in Escherichia coli strain HT115(DE)3. Transferring the Cas9-RNAi RNPs into mammalian cells achieves multidimensional genome manipulation, e.g., simultaneously knock out and knock down the target genes. Moreover, by introduction of a shRNA against the gene of human DNA ligase 4 (LIG4), significantly improved homology-directed repair was attained. Together, we develop a simple-to-use CRISPR RNP tool that has great potentials in precise genome editing, gene function analysis and gene therapy.

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qiao, j., sun, w., yin, w., ma, l., liu, y.. 2022-03-17. Rational design of Cas9 ribonucleoprotein with a "gRNA-shRNA" for multidimensional genome manipulation and enhanced homology-directed repair. https://doi.org/10.1101/2022.03.17.484717

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