CRISPR-based RNA Depletion for RNA-Seq

CRISPR-based depletion for smarter RNA-seq.

The NEXTFLEX Cas9-gRNA Depletion Enzymes identify target DNA fragments within the NGS library and introduce precise double-strand breaks in the targeted RNA transcripts. Subsequent bead-based cleanup removes cleaved molecules, enriching for intact fragments prior to PCR amplification and sequencing. The result is cleaner libraries that yield more on-target reads and deeper coverage of transcripts of interest.

Gene expression analysis by RNA sequencing (RNA-seq) is often limited by highly abundant transcripts, particularly rRNA, as well as globin mRNA and mitochondrial RNA in some sample types. These species can dominate sequencing output and reduce sensitivity for transcripts of interest, especially in low-input or complex samples. While traditional depletion methods rely on probe-based hybridization, CRISPR-based depletion using Cas9–gRNA offers a programmable and highly targeted approach to remove these transcripts. Applied after library conversion to dsDNA, this strategy selectively cleaves unwanted fragments, increasing the proportion of reads assigned to informative targets.

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Application	Revvity solution
Removal of human rRNA sequences, increasing the representation of relevant transcripts	NEXTFLEX™ Cas9-gRNA rRNA Depletion Enzyme (HMR)
Selective removal of highly expressed globin transcripts (HBA1, HBA2, HBB, & HBD), maximizing gene detection sensitivity	NEXTFLEX™ Cas9-gRNA Globin Depletion Enzyme (Human)
Removal of human mitochondrial transcripts and DNA, enabling improved recovery of nuclear-derived information in samples with high mitochondrial content	NEXTFLEX™ Cas9-gRNA Mito Depletion Enzyme (Human)

Main applications for CRISPR-based depletion

CRISPR-based RNA depletion selectively removes target transcripts, increasing the proportion of informative reads. Applied at the double-stranded DNA (dsDNA) stage, it enables precise, sequence-specific cleavage without affecting RNA handling, and integrates seamlessly into bulk and single-cell, short- and long-read RNA-seq workflows.

Bulk RNA sequencing

In whole transcriptome profiling, highly abundant RNA species can dominate reads and mask low-expression genes. CRISPR-based depletion improves transcriptome coverage by reducing rRNA and other abundant transcripts, enabling more accurate quantification and better detection of low-abundance mRNAs and non-coding RNAs. This approach is effective even with low-input samples or degraded material, such as FFPE or biofluids, where preserving RNA integrity and maximizing usable data are critical.

Single-cell RNA sequencing

Although many single-cell RNA-seq workflows include enrichment steps, abundant transcripts can still impact library complexity and sequencing efficiency, particularly in full-length or plate-based methods. Because CRISPR-based depletion is performed at the dsDNA stage, single-cell barcodes and cell-level resolution can be preserved enabling targeted depletion of dominant library fragments without affecting cell barcodes or UMI structure.

Long-read RNA sequencing

In long-read platforms, where sequencing throughput is lower and cost per read is higher, the presence of abundant RNA species can significantly reduce effective coverage. CRISPR-based depletion helps prioritize informative transcripts, enabling better characterization of isoforms, splice variants, and transcript structure while preserving RNA integrity by acting downstream at the dsDNA stage.

rRNA Depletion Enzyme (HMR)

The NEXTFLEX™ Cas9-gRNA rRNA Depletion Enzyme (HMR) targets ribosomal RNA–derived sequences from human, mouse, and rat, including both cytoplasmic (e.g., 18S and 28S) and mitochondrial rRNA species that commonly persist after library preparation.

Globin Depletion Enzyme (Human)

The NEXTFLEX™ Cas9-gRNA Globin Depletion Enzyme (Human) is designed to target highly abundant globin transcripts present in blood-derived samples, specifically hemoglobin alpha (HBA1, HBA2) and beta (HBB) genes. This targeted approach is particularly relevant for workflows involving whole blood, PBMCs, or dried blood spots.

Mito Depletion Enzyme (Human)

The NEXTFLEX™ Cas9-gRNA Mito Depletion Enzyme (Human) targets sequences derived from the human mitochondrial genome, including highly abundant mitochondrial rRNAs and mitochondrial mRNAs such as MT-CO1, MT-CO2, MT-CO3, MT-CYB, and MT-ND genes (ND1–ND6, ND4L). In addition to mitochondrial RNA–derived sequences, the enzyme also targets mitochondrial DNA (mtDNA), making it useful for applications such as ATAC-seq where mitochondrial DNA can dominate library reads.