Pan Gao1, Qing Lyu1, Amr R Ghanam1, Cicera R Lazzarotto2, Gregory A Newby3,4,5, Wei Zhang1, Mihyun Choi6, Orazio J Slivano1, Kevin Holden7, John A Walker7, Anastasia P Kadina7, Rob J Munroe8, Christian M Abratte8, John C Schimenti8, David R Liu3,4,5, Shengdar Q Tsai2, Xiaochun Long9, Joseph M Miano10. 1. Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA. 2. Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, 38195, USA. 3. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA. 4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 02138, USA. 5. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, 02138, USA. 6. Department of Physiology, Albany Medical College, Albany, NY, 12208, USA. 7. Synthego Corporation, Redwood City, CA, 94025, USA. 8. Department of Biomedical Sciences, Cornell University, Ithaca, NY, 14853, USA. 9. Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA. xlong@augusta.edu. 10. Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA. jmiano@augusta.edu.
Abstract
BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. 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 over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. 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 over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
Entities:
Keywords:
CRISPR; Gene expression; Genome editing; Mouse; Prime editing; Transcription
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