title: Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids.
abstract: Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a 'gRNA concatemer vector'. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Andersson-Rolf
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Merenda
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Roxana C
authors.initials: RC
authors.lastname: Mustata
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Taibo
authors.initials: T
authors.lastname: Li
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK.
authors.forename: Sabine
authors.initials: S
authors.lastname: Dietmann
authors.affiliation: Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK. Electronic address: bkk25@cam.ac.uk.
authors.forename: Bon-Kyoung
authors.initials: BK
authors.lastname: Koo
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D007421:Intestine, Small / Q000254:growth & development / Q000378:metabolism*; D051379:Mice; D009924:Organ Culture Techniques; D009940:Organoids / Q000254:growth & development / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D060449:Wnt Signaling Pathway / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems