# Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells

## TL;DR

This paper provides a detailed protocol for silencing retrotransposons in human stem cells using CRISPRi, with methods to validate the results.

## Contribution

A novel workflow for CRISPRi-mediated silencing of retrotransposons in hiPSCs, validated with multiome analyses.

## Key findings

- A workflow for designing gRNAs to target retrotransposons in hiPSCs is described.
- Validation procedures using RNA sequencing, CUT&RUN, and proteomics are detailed.
- The framework improves functional studies of transcriptional manipulation in stem cells.

## Abstract

Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models.

For complete details on the use and execution of this protocol, please refer to Adami et al.1

•Steps for efficient transcriptional silencing of retrotransposons•Procedure to verify on-target CRISPR-based transcriptional modifications•Analyses of CRISPR-based retrotransposon transcriptional manipulation

Steps for efficient transcriptional silencing of retrotransposons

Procedure to verify on-target CRISPR-based transcriptional modifications

Analyses of CRISPR-based retrotransposon transcriptional manipulation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models.