title: Precise and efficient scarless genome editing in stem cells using CORRECT.
abstract: 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. 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. 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. 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.
authors.affiliation: Laboratory for Brain Development and Repair, The Rockefeller University, New York, USA.
authors.forename: Dylan
authors.initials: D
authors.lastname: Kwart
authors.affiliation: Laboratory for Brain Development and Repair, The Rockefeller University, New York, USA.
authors.forename: Dominik
authors.initials: D
authors.lastname: Paquet
authors.affiliation: Laboratory for Brain Development and Repair, The Rockefeller University, New York, USA.
authors.forename: Shaun
authors.initials: S
authors.lastname: Teo
authors.affiliation: Laboratory for Brain Development and Repair, The Rockefeller University, New York, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Tessier-Lavigne
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D005434:Flow Cytometry; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D009154:Mutation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection