Program in Molecular Medicine; Graduate School of Biomedical Sciences
Amino Acids, Peptides, and Proteins | Bioinformatics | Biological Engineering | Genetics and Genomics | Molecular Biology
Here, we describe a platformed “jump board” strategy and its application in systematically engineering the essential microRNA let-7 (Fig. 1A-E) and protein coding gene lin-28 (Fig. 1F) in C. elegans. We chose the jump board protospacer sequence (INPP4A) which is (1) comprised of a PAM site and a protospacer antisense to a crRNA with experimentally confirmed high editing efficiency (INPP4A-crRNA), and (2) non-homologous to C. elegans genome, including the genetic balancer we used (mnDp1). Notably, the jump board protospacer contains an EcoRV restriction site, which can be utilized for rapid large-scale genotyping by which HDR events can be identified in the F1 generation (Fig. 1C). Using the jump board strategy, we have so far created 28 let-7 alleles for various experimental purposes, among which 15 alleles showed lethality and require rescue by mnDp1. Note that the let-7 jump board allele (ma393) itself is a new let-7 null allele in which the precursor-let-7 is completely removed.
CRISPR/Cas9 genome editing
Rights and Permissions
Copyright: © 2020 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
DOI of Published Version
Duan Y, Choi S, Nelson C, Ambros V. Engineering essential genes with a "jump board" strategy using CRISPR/Cas9. MicroPubl Biol. 2020 Oct 8;2020:10.17912/micropub.biology.000315. doi: 10.17912/micropub.biology.000315. PMID: 33274316; PMCID: PMC7704125. Link to article on publisher's site
Duan Y, Choi S, Nelson C, Ambros VR. (2020). Engineering essential genes with a "jump board" strategy using CRISPR/Cas9. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.17912/micropub.biology.000315. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1900
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.