Using CAPTURE to detect spacer acquisition in native CRISPR arrays.
Nat Protoc
; 14(3): 976-990, 2019 03.
Article
en En
| MEDLINE
| ID: mdl-30742049
ABSTRACT
CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 105 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa
/
Sistemas CRISPR-Cas
Idioma:
En
Revista:
Nat Protoc
Año:
2019
Tipo del documento:
Article
País de afiliación:
Países Bajos