Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
Genome Biol
; 22(1): 83, 2021 03 16.
Article
em En
| MEDLINE
| ID: mdl-33722289
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.Palavras-chave
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Base de dados:
MEDLINE
Assunto principal:
Regulação da Expressão Gênica
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Mutação Puntual
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Sistemas CRISPR-Cas
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Edição de Genes
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article