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Wettengel, J.M. ; Hansen-Palmus, L. ; Yusova, S.* ; Rust, L.* ; Biswas, S.* ; Carson, J.* ; Ryu, J.* ; Bimber, B.N.* ; Hennebold, J.D.* ; Burwitz, B.J.*

A multifunctional and highly adaptable reporter system for CRISPR/Cas editing.

Int. J. Mol. Sci. 24:13 (2023)
Publ. Version/Full Text DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Crispr-cas Reporter System ; Cas9 Activity ; Hdr ; Nhej ; Ascas12a ; Sacas9 ; Spcas9; Read Alignment; Dna; Endonuclease; Enrichment
ISSN (print) / ISBN 1422-0067
e-ISSN 1661-6596
Journal International Journal of Molecular Sciences
Quellenangaben Volume: 24, Issue: 9, Pages: , Article Number: 13 Supplement: ,
Publisher MDPI
Publishing Place Basel
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Virology (VIRO)
Grants Deutscher Akademischer Austauschdienst
amfAR
National Institutes of Health