| First Author | Cassidy AM | Year | 2022 |
| Journal | Heliyon | Volume | 8 |
| Issue | 12 | Pages | e12630 |
| PubMed ID | 36619446 | Mgi Jnum | J:342085 |
| Mgi Id | MGI:7545662 | Doi | 10.1016/j.heliyon.2022.e12630 |
| Citation | Cassidy AM, et al. (2022) One-step generation of a conditional allele in mice using a short artificial intron. Heliyon 8(12):e12630 |
| abstractText | Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice. |
