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1.
Nucleic Acids Res ; 47(14): 7402-7417, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127293

RESUMO

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.


Assuntos
Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos de Mamíferos/genética , Edição de Genes/métodos , Deleção de Sequência , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo , Cromossomos de Mamíferos/metabolismo , Reparo do DNA por Junção de Extremidades , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Camundongos , Modelos Genéticos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
2.
Nat Commun ; 13(1): 773, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-35140205

RESUMO

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.


Assuntos
Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Expressão Gênica , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , DNA/química , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Mesoderma/metabolismo , Camundongos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas
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