Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis.
Nucleic Acids Res
; 46(12): 5950-5966, 2018 07 06.
Article
en En
| MEDLINE
| ID: mdl-29860520
ABSTRACT
Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Oligonucleótidos
/
Transcripción Genética
/
Oligonucleótidos Antisentido
/
Interferencia de ARN
/
Técnicas de Silenciamiento del Gen
/
Sistemas CRISPR-Cas
Tipo de estudio:
Evaluation_studies
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2018
Tipo del documento:
Article
País de afiliación:
Reino Unido