RESUMO
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a CâT (or GâA) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Citidina/genética , Engenharia Genética/métodos , Genoma/genética , Mutação Puntual/genética , Uridina/genética , Desaminase APOBEC-1 , Animais , Apolipoproteína E4/genética , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , DNA/metabolismo , Clivagem do DNA , Reparo do DNA , Desoxirribonuclease I/metabolismo , Genes p53/genética , Humanos , Mutação INDEL/genética , Camundongos , RNA Guia de Cinetoplastídeos/genética , Moldes Genéticos , Uracila-DNA Glicosidase/antagonistas & inibidoresRESUMO
Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library.
Assuntos
Proteínas de Ligação a DNA/genética , Engenharia de Proteínas , Transcrição Gênica , Proteínas de Ligação a DNA/química , Escherichia coli/genética , LuzRESUMO
A tandem ring-closing metathesis reaction using ruthenium catalyst was carried out to synthesize various fused bicyclic compounds containing both small and large rings. Fast ring-closure of the small ring and slow ring-closure of the large ring resulted in the formation of only one isomer. Further manipulation such as the Diels-Alder reaction was carried out to prepare a complex molecule containing multiple rings of different sizes.
