Two-step CRISPR-Cas9 protocol for transposable element deletion in D. melanogaster natural populations

• Published in: STAR protocols Vol. 4; no. 3; p. 102501
• Main Authors: Merenciano, Miriam, Aguilera, Laura, González, Josefa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2023; Elsevier
• Subjects: Animals; CRISPR; CRISPR-Cas Systems - genetics; DNA Transposable Elements - genetics; Drosophila melanogaster - genetics; Evolutionary Biology; Genetics; Model Organisms; Molecular Biology; Protocol; Recombination, Genetic; RNA, Guide, CRISPR-Cas Systems; CRISPR; Genetics; Model Organisms; Molecular Biology; Evolutionary Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102501

We present a protocol for generating a precise deletion, without altering the genetic background of the strain, of a transposable element (TE) in a natural population of Drosophila melanogaster using two steps of CRISPR-Cas9 homology-directed repair. We describe steps for replacing the TE by a fluorescent marker and for subsequent marker removal using single-guide RNAs, repair plasmids, and microinjection. We also detail steps for screening the deletion of the TE and generating a homozygous mutant strain. For complete details on the use and execution of this protocol, please refer to Merenciano and Gonzalez.1 [Display omitted] •Generation of a precise deletion without altering the genetic background of the strain•The protocol allows for the visual screening of mutants in the two CRISPR editing steps•Deletion of a transposable element to study its functional impact in the genome Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We present a protocol for generating a precise deletion, without altering the genetic background of the strain, of a transposable element (TE) in a natural population of Drosophila melanogaster using two steps of CRISPR-Cas9 homology-directed repair. We describe steps for replacing the TE by a fluorescent marker and for subsequent marker removal using single-guide RNAs, repair plasmids, and microinjection. We also detail steps for screening the deletion of the TE and generating a homozygous mutant strain.