RESUMO
SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Camundongos , Animais , Humanos , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Streptococcus pyogenes , Edição de Genes , MutagêneseRESUMO
CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.
Assuntos
Sistemas CRISPR-Cas , Escherichia coli , Edição de Genes , Genoma Bacteriano , Edição de Genes/métodos , Escherichia coli/genética , RNA Guia de Sistemas CRISPR-Cas/genética , Temperatura Baixa , TemperaturaRESUMO
The clustered regularly interspaced short palindromic repeats and associated protein (CRISPR-Cas) toolbox enables targeted mutations to be introduced into a genome. However, the delivery of appropriately sized Cas effectors to develop transgene-free edited plants is a limiting factor. A novel mini CRISPR-Cas12f1 system recently reported by Wu et al. overcomes this challenge by deploying viral-based vectors and nanoparticles (NPs) as carriers.