High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products.
Dev Biol
; 484: 22-29, 2022 04.
Article
en En
| MEDLINE
| ID: mdl-35149003
ABSTRACT
Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels-down to 2 base pairs-can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Pez Cebra
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
Límite:
Animals
Idioma:
En
Revista:
Dev Biol
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos