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
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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. UMass Chan Medical School Faculty Publications. https://doi.org/10.17912/micropub.biology.000315. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1900
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This work is licensed under a Creative Commons Attribution 4.0 License.