Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Gao, P.; Lyu, Q.; Ghanam, A. R.; Lazzarotto, C. R.; Newby, G. A.; Zhang, W.; Choi, M.; Slivano, O. J.; Holden, K.; Walker, J. A.; Kadina, A. P.; Munroe, R. J.; Abratte, C. M.; Schimenti, J. C.; Liu, D. R.; Tsai, S. Q.; Miano, J. M.

doi:10.1101/2020.11.07.372748

bioRxiv · 10.1101/2020.11.07.372748

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Gao, P.·Lyu, Q.·Ghanam, A. R.·Lazzarotto, C. R.·Newby, G. A.·Zhang, W.·Choi, M.·Slivano, O. J.·Holden, K.·Walker, J. A.·Kadina, A. P.·Munroe, R. J.·Abratte, C. M.·Schimenti, J. C.·Liu, D. R.·Tsai, S. Q.·Miano, J. M.

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Abstract

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

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Gao, P., Lyu, Q., Ghanam, A. R., Lazzarotto, C. R., Newby, G. A., Zhang, W., Choi, M., Slivano, O. J., Holden, K., Walker, J. A., Kadina, A. P., Munroe, R. J., Abratte, C. M., Schimenti, J. C., Liu, D. R., Tsai, S. Q., Miano, J. M.. 2020-11-08. Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression. https://doi.org/10.1101/2020.11.07.372748

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