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The majority of HDR protocols are based on plasmid-based expression of Cas9 and the gene-specific guide RNAs. HDR edits are a common occurrence in Caenorhabditis elegans' gonad. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA trans-activating crRNA ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high rate. We developed an entirely cloning-free scheme for the generation of seamless HDR edits without selection, building on our earlier finding that PCR fragments with 35-base homology are effective repair templates.
Source link: https://doi.org/10.1534/genetics.115.179382
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