Tutorial: design and execution of CRISPR in vivo screens.

Autor: Braun CJ; Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich, Germany. chr.braun@med.lmu.de.; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany. chr.braun@med.lmu.de.; Hopp Children's Cancer Center Heidelberg (KiTZ), German Cancer Research Center (DKFZ), Heidelberg, Germany. chr.braun@med.lmu.de., Adames AC; Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich, Germany., Saur D; Institute of Experimental Cancer Therapy, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany., Rad R; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. roland.rad@tum.de.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2022 Sep; Vol. 17 (9), pp. 1903-1925. Date of Electronic Publication: 2022 Jul 15.
DOI: 10.1038/s41596-022-00700-y
Abstrakt: Here we provide a detailed tutorial on CRISPR in vivo screening. Using the mouse as the model organism, we introduce a range of CRISPR tools and applications, delineate general considerations for 'transplantation-based' or 'direct in vivo' screening design, and provide details on technical execution, sequencing readouts, computational analyses and data interpretation. In vivo screens face unique pitfalls and limitations, such as delivery issues or library bottlenecking, which must be counteracted to avoid screening failure or flawed conclusions. A broad variety of in vivo phenotypes can be interrogated such as organ development, hematopoietic lineage decision and evolutionary licensing in oncogenesis. We describe experimental strategies to address various biological questions and provide an outlook on emerging CRISPR applications, such as genetic interaction screening. These technological advances create potent new opportunities to dissect the molecular underpinnings of complex organismal phenotypes.
(© 2022. Springer Nature Limited.)
Databáze: MEDLINE
Externí odkaz: