Epigenetic silencing is a barrier to editing the X chromosome using the SEC-based CRISPR/Cas9 knock-in protocol in C. elegans

TL;DR

Scientists found that editing the X chromosome in C. elegans using a CRISPR method is blocked by epigenetic silencing, but they developed a workaround using RNAi to temporarily reduce this silencing.

Contribution

The study identifies epigenetic silencing as a barrier to SEC-based CRISPR/Cas9 knock-in on the X chromosome and proposes a PRC2 RNAi-based solution.

Findings

SEC-based CRISPR/Cas9 knock-in fails on the X chromosome due to epigenetic silencing in primordial germ cells.

PRC2 RNAi temporarily reduces silencing, enabling successful SEC floxing on the X chromosome.

The workaround preserves germ line development while allowing X chromosome tagging.

Abstract

Self-excising-cassette (SEC)-based CRISPR/Cas9 knock-in is widely used for generating endogenous fluorescent protein tags in

C. elegans

. Here, we report a lack of success targeting the X chromosome using this method. CRISPR/Cas9 works as intended, but subsequent floxing of the SEC is blocked. Given that the X chromosome is epigenetically silenced in primordial germ cells (PGCs), this is a logical result. To circumvent this barrier, we suppressed polycomb repressive complex 2 (PRC2) with RNAi to transiently and reversibly reduce silencing in the PGCs, creating a brief window where the X chromosome is amenable to floxing without compromising germ line development. Overall, our results reveal a previously unrecognized limitation of SEC-based CRISPR/Cas9 knock-in and identify a reliable workaround for tagging proteins encoded on the X chromosome.

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