Adding introns to Cas9 expression constructs reduced and improved target-gene activation in cell lines.
Evidence
This was a cell-line genetic construct study testing multiple Cas9 and CRISPRa expression cassettes, intron sequences, and chromatin opening elements.
Caveat
The findings are limited to engineered cell systems and construct-level expression outcomes rather than in vivo editing performance or long-term therapeutic use.
Simplified
Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.
Key numbers
11.6×
Increase in dCas9-activator expression
Transcript abundance of SAM-dCas9-NFZ with IVS2 vs. intronless construct.
15.4×
Increased activation of CD2 expression
Proportion of cells expressing CD2 with intron-containing dCas9 vs. intronless dCas9.
< 0.0009
Reduction in Cas9-BFP silencing
Comparison of silencing rates in cells with and without the IVS2 intron.
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