Abstract
Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA.
Highlights
CRISPR/Cas is a bacterial/archaeal immune system that can be adapted to perform sequence-specific genome engineering in mammalian cells and to make novel model organisms [1]
Novel genetic material can be inserted at the cleavage site by supplying a DNA template, which can be as simple as single-base mutagenesis or insertion of kilobases of new DNA content via homology-directed repair (HDR)
Cells were lysed 48 h after transfection and DNA was tested for genome editing at the HPRT1 locus using a T7 Endonuclease I (T7EI) Endonuclease mismatch cleavage (EMC) assay and for insertion of the EcoRI HDR template by EcoRI digestion
Summary
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) is a bacterial/archaeal immune system that can be adapted to perform sequence-specific genome engineering in mammalian cells and to make novel model organisms [1]. The guide RNA provides sequence specificity and targets the Cas nuclease to a complementary site in the genome where the nuclease creates a double-stranded break. The double-stranded break is healed by cellular repair machinery (non-homologous end joining, or NHEJ), which is often imprecise and can disrupt the amino acid coding sequence if the guide targets a coding exon. The guide RNA occurs naturally as a 2-molecule complex comprising a target-specific crisprRNA (crRNA) bound to a trans-activating crRNA (tracrRNA) that directs binding of the RNAs to Cas. Novel genetic material can be inserted at the cleavage site by supplying a DNA template, which can be as simple as single-base mutagenesis or insertion of kilobases of new DNA content via homology-directed repair (HDR)
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