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The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously.Deletion or insertion of large DNA fragments through direct micromanipulation of one-cell embryos via Cas9 is challenging.Through the injection of Cas9 protein instead of mRNA into embryos,off-target effects of Cas9 were reduced and point mutation knockin efficiency were increased.Large genomic DNA fragment (up to 95Kb) deletion mice were generated for in vivo study of lncRNAs and formyl peptide receptors.Through site-specific insertion of a 2.7kb CreERT2 cassette into the mouse Nfatc1 locus, hair follicle stem cells were labeled and traced.In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.