Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.
Genome Biol
; 25(1): 131, 2024 05 21.
Article
em En
| MEDLINE
| ID: mdl-38773623
ABSTRACT
BACKGROUND:
High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites.RESULTS:
We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired.CONCLUSIONS:
This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Plantas
/
Oryza
/
Edição de Genes
Idioma:
En
Revista:
Genome Biol
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA
Ano de publicação:
2024
Tipo de documento:
Article
País de publicação:
Reino Unido