Jun 17, 2026
CRISPRa Perturb-Seq Screen
- Julian Pulecio1,
- Michael Beer2,
- Danwei Huangfu1
- 1Sloan Kettering Institute;
- 2Johns Hopkins University
Protocol Citation: Julian Pulecio, Michael Beer, Danwei Huangfu 2026. CRISPRa Perturb-Seq Screen. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2xme4v1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 31, 2026
Last Modified: June 17, 2026
Protocol Integer ID: 318243
Keywords: CRISPRa, Perturb-Seq, 10x Genomics, CROP-seq, Competent Chromatin Regions, OCT4, NANOG, TET1, QSER1, H3K27me3, CAGE-seq, FANTOM5, Human Embryonic Stem Cell, crispr activation, competent chromatin region, human embryonic stem cell, embryonic stem cell, lineage gene, crispra perturb, crispra, seq approach in human, seq screen
Funders Acknowledgements:
NHGRI
Grant ID: U01 HG012051
Abstract
To identify and validate competent chromatin regions (CCRs) of lineage gene using a CRISPR activation (CRISPRa) Perturb-Seq approach in human embryonic stem cells (hESCs).
Region identification and gRNA library design
We tested whether the regions with enriched binding of the pluripotent TFs OCT4 and NANOG (ON) alone or together with the enriched binding of QSER1 and TET1 (ONQT) could predict the presence of CCRs associated with lineage genes at a large genome scale.
We first identified lineage TFs that are lowly or not expressed in hESC according to our previous RNA-seq data3 and selected 70 TFs distributed across 18 chromosomes.
We verified that these TFs become active in fetal and adult tissues from the three germ layers. The data used to identify the gene expression levels of the lineage TFs in adult tissues were obtained from the GTEx Portal v10 release (https://www.gtexportal.org/).
We determined the TADs called in hESCs where those TF loci are located and looked for regions containing the ON or ONQT signatures by overlapping called peaks from previous ChIP-seq experiments using the findOverlaps functions from the gRanges package in R.
In addition, we sought control regions associated with poised enhancers that contain H3K27me3 peaks but did not contain any of the ONQT factors (Table S9).
The final selected regions were annotated with the annotatePeak function from the ChIPseeker package in R, and only the regions defined as interdistal or intronic with a total width less than 1kb were selected (candidate enhancer regions).
A total of 80 ON, 78 ONQT, and 76 H3K27me3 regions were selected for the library.
We selected the gRNAs targeting our candidate regions through guidescan2 (https://guidescan.com/) and, when possible, selected the top 4 gRNAs per region.
In addition, as positive controls to validate the functionality of our CRISPRa tool in the 10x genomics assays, we identified the promoter regions of 35 lineage TFs and designed 2 gRNAs per promoter based on their transcriptional starting site defined by the CAGE-seq FANTOM5 database (https://fantom.gsc.riken.jp/5/).
Finally, a second small gRNA library was designed with 15 gRNAs targeting safe-harbor regions and non-targeting sequences previously validated. All gRNA sequences and region coordinates are included in Table S7 (Pulecio et al., 2026).
See the supplemental material of Pulecio 2026 Functional chromatin signatures premark future lineage-specific enhancers.
gRNA library cloning and transduction
sgRNA were synthesized on-Chip (Agilent) and cloned into the MS2-CROP backbone by the MSKCC Gene Editing & Screening Core Facility (CROP-sgRNA-MS2 was a gift from Wolf Reik - Addgene #153457).
In brief, this backbone contains a U6 promoter that drives the expression of a single MS2-gRNA and an EF-1 promoter that drives the transcription of a cassette that encloses mCherry-2a-Puromycin selection markers and the gRNA sequence at its 3’ end.
This dual system allows the identification of the gRNA sequences in the transcriptome at a single-cell level.
In parallel, oligos from the negative control gRNA library were synthesized (EtOn Bio) and cloned into the MS2-CROP backbone, to generate a small "control gRNA library" to facilitate the detection of cells transduced with control gRNAs for the downstream 10x experiments.
See the Method section of Pulecio 2026 Functional chromatin signatures premark future lineage-specific enhancers.
gRNAs lentiviral libraries were prepared as described in "Lentiviral gRNA libraries" and titered at different concentrations that were subsequently measured, based on viral RNA levels detected in transduced cells.
See the Method section of Pulecio 2026 Functional chromatin signatures premark future lineage-specific enhancers.
Libraries at MOI ~1.0 (gRNAs/cell) and MOI ~3 (gRNAs/cell) were selected to represent single and multiplexed conditions.
hESC iSAM cells were independently transduced at high and low MOIs with the libraries of the candidate CCRs + promoters and the "control gRNA libraries" (4 independent transductions).
See the Method section of Pulecio 2026 Functional chromatin signatures premark future lineage-specific enhancers.
After selection for 4 days with puromycin, cells were treated with doxycycline for 3 additional days.
Single-cell 10x genomics
After doxycycline-induced activation of the iSAM CRISPRa tool for 3 days, cells were washed with PBS and treated with TrypLE Select for 2 minutes, washed 2 times with PBS containing 0.04% BSA, and filtered through CellTrics (20μm, Sysmex) to be sorted in a Flow Cytometer, using the mCherry live marker that distinguishes the transduced cell population expressing the cassette that contains the gRNA sequences (see Addgene #153457).
mCherry+ hESCs transduced with the 4 different library conditions were FACS-sorted, and 2 cellular mixes were prepared as follows: 1.) 500000 cells at a 97:3 ratio = low MOI candidate CCR-library transduced hESCs: low MOI negative control transduced hESCs. 2.) 100000 cells at a 97:3 ratio = high MOI candidate CCR-library transduced hESCs: high MOI negative control transduced hESCs.
Cellular suspensions were loaded on a Chromium Controller targeting 30000 cells per reaction.
Seven reactions for the low MOI condition and one reaction for the high MOI condition were loaded.
Single-cell 3′ transcriptome libraries were generated following the manufacturer’s instructions (10x Genomics Chromium Single Cell 3′ Reagent Kit v3.1-Dual Index User Guide).
gRNA libraries were generated by amplifying the fragment that contains the gRNA sequence from the MS2-CROP construct (see oligos in Table S8), and a second PCR to add index adaptors from the Chromium Single Cell 3′ Reagent Kit v3.1-Dual Index.
See the supplemental material of Pulecio 2026 Functional chromatin signatures premark future lineage-specific enhancers.
Libraries were then quantified on the Agilent Bioanalyzer with a high-sensitivity chip (Agilent), and Kapa DNA quantification kit for Illumina platforms (Roche).
The libraries were sequenced on a NovaX platform following the manufacturer’s guidelines.
The total number of reads was adjusted to aim for ~20000 average reads per cell. (~7x10^9 total reads for the seven low MOI transcriptome reactions and 4.5x10^8 total reads for the high MOI transcriptome reaction).
Protocol references
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