Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Inducible in vivo genome editing with CRISPR-Cas9. A Cas9-guide RNA complex preorganized for target DNA recognition. Self versus non-self discrimination during CRISPR RNA-directed immunity. CRISPR/Cas, the immune system of bacteria and archaea. Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Multiplex genome engineering using CRISPR/Cas systems. RNA-guided human genome engineering via Cas9. RNA-programmed genome editing in human cells. Development and applications of CRISPR-Cas9 for genome engineering. #MACVECTOR V12 DOWNLOAD HOW TO#This protocol outlines in detail how to implement either the re-Guide or re-Cas variants of CORRECT to generate scarlessly edited isogenic stem cell lines with intended monoallelic and biallelic sequence changes in ∼3 months. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells.
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