Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.
CRISPR J
; 6(2): 176-182, 2023 04.
Article
em En
| MEDLINE
| ID: mdl-37071670
ABSTRACT
The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps:
(1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
Edição de Genes
Idioma:
En
Revista:
CRISPR J
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos