Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.
Nat Biotechnol
; 41(7): 968-979, 2023 Jul.
Article
en En
| MEDLINE
| ID: mdl-36593413
ABSTRACT
CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5' nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Elementos Transponibles de ADN
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
Tipo de estudio:
Prognostic_studies
/
Risk_factors_studies
Límite:
Humans
Idioma:
En
Revista:
Nat Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos