title: A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair.
abstract: The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using thePiggybactransposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e.astable, butreversible,integratedreporter forassayingCRISPR/Cas-stimulatedHDRactivity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactivePiggybactransposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors.forename: Yahong
authors.initials: Y
authors.lastname: Wen
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors.forename: Grace
authors.initials: G
authors.lastname: Liao
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pritchard
authors.affiliation: First Hospital of China Medical University, Department of Breast Surgery, Shenyang, China 110001.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Zhao
authors.affiliation: Genome Engineering and iPSC Center (GEiC), Department of Genetics, Washington University, Saint Louis, Missouri 63110, and.
authors.forename: Jon P
authors.initials: JP
authors.lastname: Connelly
authors.affiliation: Genome Engineering and iPSC Center (GEiC), Department of Genetics, Washington University, Saint Louis, Missouri 63110, and.
authors.forename: Shondra M
authors.initials: SM
authors.lastname: Pruett-Miller
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors.forename: Valerie
authors.initials: V
authors.lastname: Blanc
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110.
authors.forename: Nicholas O
authors.initials: NO
authors.lastname: Davidson
authors.affiliation: From the Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri 63110, bmadison@wustl.edu.
authors.forename: Blair B
authors.identifier: 0000-0001-7457-1832
authors.initials: BB
authors.lastname: Madison
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice
keywords: CRISPR/Cas; DNA endonuclease; HDR; Piggybac; gene knockout; genome editing; homologous recombination; surrogate reporter; transposable element (TE)