NEXTFLEX Cas9-gRNA Mito Depletion Enzyme (Human)

CRISPR-based depletion uses a Cas9–guide RNA complex to recognize and cut molecules derived from the mitochondrial genome in already-prepared DNA- and RNA-derived dsDNA libraries. Removing these highly abundant fragments reduces the fraction of reads that land on mtDNA and mitochondrial transcripts, so more of your fixed sequencing budget is spent on nuclear genes, regulatory elements, and other loci of interest. Because depletion happens after conversion to double-stranded DNA, it also helps preserve material from low-input or partially degraded samples that might be lost with earlier, RNA-level depletion steps.

Working at the dsDNA stage avoids repeated handling of fragile RNA and lets you treat the library only after you know it has high mitochondrial content. Traditional strategies that target RNA or genomic DNA can be sensitive to RNA integrity, extraction method, or cell type, and they can risk losing rare transcripts or introducing unwanted bias. Applying depletion to dsDNA libraries instead allows you to use your preferred extraction and library prep protocol, then remove mitochondrial fragments with a single incubate-and-clean-up step that slots into existing workflows.

The NEXTFLEX Cas9-gRNA Mito Depletion Enzyme (Human) is programmed specifically against human mitochondrial DNA and selected human nuclear mitochondrial pseudogenes. It is intended for human samples only and has been designed for use with a wide range of human library types, including ATAC-seq, low-pass or whole-genome DNA-seq, targeted panels, bulk RNA-seq, and single-cell or single-nucleus workflows once material has been converted to double-stranded DNA.

Because depletion occurs on double-stranded DNA, this module is broadly compatible with most human DNA-seq and RNA-seq library prep kits, including NEXTFLEX Rapid XP v2 DNA-seq kits, NEXTFLEX Rapid Directional RNA-seq kits, and other commercial or in-house workflows that produce dsDNA libraries. It can be used ahead of short-read or long-read sequencing, and it is suitable for both manual and automated workflows, as long as you can introduce a one-hour incubation and a subsequent cleanup step.

Yes. Because the depletion step is performed on dsDNA rather than on RNA, the module is well suited to low-input or partially degraded RNA or DNA samples that can still be converted into sequenceable libraries. The Cas9–gRNA complex acts on library fragments regardless of the original RNA integrity, so you can combine challenging samples with standard RNA-seq or DNA-seq workflows and then remove mitochondrial background at the library stage. As with any low-input application, we recommend monitoring library complexity and standard QC metrics.

In internal testing on human libraries, treatment with the NEXTFLEX Cas9-gRNA Mito Depletion Enzyme (Human) has reduced mitochondrial reads from roughly 9% of total reads to well under 1%, corresponding to more than a 98% drop in mitochondrial fraction under recommended conditions. Actual depletion will depend on input amount, library design, tissue type, and the initial mitochondrial burden, so users may need to optimize enzyme-to-DNA ratios for their specific samples, especially when working at higher inputs.

The guide RNA pool contains 657 unique guides targeting 17 mtDNA-encoded genes together with selected nuclear mitochondrial pseudogenes. This guide RNA pool was designed to avoid protein-coding nuclear genes outside of those pseudogene loci. As a result, the primary effect of the enzyme is to remove molecules derived from the mitochondrial genome while preserving nuclear content. As with any targeted depletion method, we recommend validating performance and checking for unexpected effects when working with unusual library designs, extreme GC content, or highly customized workflows.

Yes. This product is a ready-to-use Cas9–guide RNA complex for DASH-style (Depletion of Abundant Sequences by Hybridization) removal of mitochondrial genome–derived fragments from human dsDNA libraries. It can be positioned before or after PCR, depending on your workflow, and can in principle be combined with other Cas9-based depletion modules that target different sequences, provided that reaction conditions and cleanup steps are carefully optimized so that each module performs as expected.

Yes, as long as the material has been converted into a dsDNA library or amplified cDNA pool. The enzyme can be applied to human single-cell or single-nucleus ATAC-seq, RNA-seq, or multiome libraries to reduce mitochondrial reads that inflate mitochondrial QC metrics and consume sequencing depth. When using the module in single-cell workflows, it is important to interpret mitochondrial read percentages considering the depletion step and to confirm that cell calling and clustering behavior remain consistent with expectations.

No. The NEXTFLEX Cas9-gRNA Mito Depletion Enzyme (Human) is designed to remove sequences derived from the mitochondrial genome. It is ideal for experiments where mitochondrial signal is a source of background noise, and the focus is on nuclear features. If your primary goal is to profile mtDNA variants, copy number, or heteroplasmy, you should not use this depletion module, or you should test substantially reduced enzyme levels to ensure that the mitochondrial reads you need are preserved.