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rhAmpSeq™ CRISPR Library Kit

NGS-ready amplicon libraries. Fast. Easy. Convenient.

Our proprietary rhAmp™ PCR technology delivers optimal amplification with just two PCR reactions and minimal hands-on time. Combine the rhAmpSeq Library Kit with rhAmpSeq CRISPR Panels and rhAmpSeq Index Primers for rapid preparation of amplicon libraries for sequencing on Illumina platforms.

The rhAmpSeq CRISPR Library Kit comes with Analysis Credits for the rhAmpSeq CRISPR Analysis Tool, a cloud-based software package for quantification of CRISPR editing events. This tool generates publication-ready editing reports without the need for advanced bioinformatics skills.

  • Sequencing-ready amplicon libraries for analyzing CRISPR on- and off-target editing results
  • Low-input, uniform amplification for hundreds of target sites
  • High coverage uniformity and high on-target rate

Ordering

To complete your rhAmpSeq CRISPR workflow, you will also need a rhAmpSeq CRISPR Panel and rhAmpSeq Index Primers.

Analysis Credits are provided with every rhAmpSeq CRISPR Library Kit purchased allowing you to use the rhAmpSeq CRISPR Analysis Tool. Each credit allows for the analysis of up to 500 targets for one, indexed sample. Upon purchase of this product, your activation code to redeem for Analysis Credits is emailed directly to you.

* This product is currently not available in China or Russia.

The rhAmpSeq CRISPR Library Kit contains two amplification mixes optimized for rapid preparation of highly specific, sequencing-ready amplicon libraries.

  • rhAmpSeq Library Mix 1—optimized for high specificity and multiplexing capability. This mix is for Targeted rhAmp PCR 1 in the rhAmpSeq workflow (Figure 1).
  • rhAmpSeq Library Mix 2—optimized for high-efficiency amplification. This mix is for Indexing PCR 2 in the rhAmpSeq workflow. In this universal PCR step, rhAmpSeq Index Primers add index barcodes and Illumina P5/P7 adapter sequences to the library (Figure 1).

Harnessing the power of rhAmp PCR

Our proprietary rhAmp PCR technology drives the rhAmpSeq CRISPR Analysis System. This technology harnesses the intrinsic properties of the RNase H2 enzyme and RNA-base–containing blocked primers (rhAmp primers), minimizing primer dimers and enabling rhAmpSeq panels to be highly multiplexed.

Those two advantages—minimal primer dimers and high multiplexing capabilities—allowed us to develop the high-performance rhAmpSeq CRISPR Analysis System, whose fast, easy workflow requires only two PCR amplification steps (Figure 1) to generate amplicon libraries for Illumina platform sequencing.

Figure 1. Detail of amplification steps in the rhAmpSeq workflow. RNase H2 activates rhAmp primers by target-specific cleavage of the RNA base within the DNA:RNA duplex, removing a 3′ blocker. RNase H2 activity is highly specific, thus reducing the amount of amplification from non-specific hybridization and primer dimers. Only activated rhAmp primers can be extended to generate target amplicons.

Illumina sample barcodes and P5/P7 sequences are incorporated during Indexing PCR 2.

Technical details

Because all rhAmpSeq reagents are compatible with both our regular and high-throughput library preparation protocols, you can choose the best workflow for each experiment without having to buy different reagents. Table 1 shows the features and specifications common to both rhAmpSeq system protocols.

Table 1. rhAmpSeq system features and specifications.

FeatureSpecification
Supported applications/protocolsOn- and off-target analysis of genome editing experiments
High-throughput analysis of on-target genome editing experiments
Insert sizeFlexible (50–200 nt)
Custom panel sizeUp to 5000 amplicons per panel
Sample indexing capability96 index sequences (up to 9216 combinations)
Compatible platformsIllumina
Hands-on time*1–1.5 hr
Data analysis time**0.5–2 hr

* Estimated time to process 12–96 samples using manual pipetting, including reaction setup, cleanup, library quantification, and normalization steps
** Estimated time to process sequencing data (up to 500 amplicons) through rhAmpSeq CRISPR Analysis Tool

Robust on-target rate, uniform coverage with no primer-dimer amplification

The rhAmpSeq CRISPR Analysis System enables singleplex or multiplex analysis of CRISPR edits by next-generation sequencing (NGS) in less than a week. Our custom design tool ensures maximal compatibility between the target primers and the rhAmpSeq CRISPR Library Kit prevents primer-dimer amplification during the two simple PCR steps required for sequencing on any Illumina platform. The system provides access to an intuitive data analysis tool that generates publication-quality figures without the need for advanced bioinformatics expertise.

Figure 2. The rhAmpSeq CRISPR Analysis System provides robust on-target rate and uniform coverage with no primer-dimer amplicons. (A) The workflow for targeted enrichment via amplification with the rhAmpSeq CRISPR Analysis System generates NGS-ready libraries in two straightforward PCR steps. (B) The rhAmpSeq CRISPR on-target rate represents the fraction of reads from expected targets in the rhAmpSeq CRISPR Panel relative to all mapped reads from the library. In this experiment, genomic DNA samples (Coriell) were amplified with a 19-, 282-, and 994-plex rhAmpSeq CRISPR Panel with 10 and 50 ng of input DNA. All panels were highly specific, giving on-target mapping rates of >98%. (C) The uniformity of target amplification in a 990-plex is shown. In this experiment, 95% of all targets give coverage that is >0.2X of the mean coverage depth of the entire panel. (D) DNA-only primers compared to rhAmp primers show an accumulation of primer dimers in this 35-plex reaction with unblocked DNA primers, while no primer-dimers are observed when using end-blocked, RNaseH2-cleavable, rhAmp primers.

Panel uniformity

High uniformity and specificity with rhAmpSeq CRISPR Panels allow for interrogation of on- and off-target editing in a single library prep with PCR amplification as represented below using the popular AAVS1 “safe harbor” site (Figure 3). All 28 empirically identified off-target sites can be verified when Cas9 is constitutively expressed in HEK293 cells. Using ribonucleoprotein (RNP) delivery of the Cas9/guide complex in conjunction with Alt-R™ S.p. HiFi Cas9 Nuclease V3 dramatically reduces off-target editing.

Figure 3. High uniformity and specificity with rhAmpSeq CRISPR Panels allow for interrogation of on- and off-target editing in a single library prep with PCR amplification. HEK293 cells constitutively expressing S.p. Cas9 nuclease were electroporated with 10 µM AAVS1-targeting Alt-R sgRNA. Alternatively, standard HEK293 cells were electroporated with 4 µM Alt-R wild-type (WT) or HiFi Cas9 Nuclease complexed to the AAVS1 sgRNA (at a 1:1.2 protein to gRNA ratio), including 4 µM Alt-R Cas9 Electroporation Enhancer using the Amaxa™ Nucleofector™ 96-well Shuttle™ System (Lonza). gDNA was isolated and amplified using a custom rhAmpSeq CRISPR Panel containing amplicons for the on-target and 28 of the top off-target sites identified by GUIDE-Seq. Amplicon sequencing on the Illumina MiSeq (v2 chemistry, 150 bp paired-end reads) was performed and analyzed using the rhAmpSeq CRISPR Analysis Tool. (A) The histogram of panel coverage shows 100% of assays have read coverage depth that is >0.2X of the mean read coverage depth for all assays in the panel, indicating highly uniform enrichment via amplification. (B) Fragment analysis of final panels in duplicate shows the expected fragment sizes of 300–400 bp with no primer dimers present. (C) NHEJ editing for the on-target locus (Assay 1) and off-target loci (Assays 2-29) is reported using an untreated control for background subtraction. Background editing was <0.2% for all assays in this panel.

Batch analysis of single amplicon assays

While the rhAmpSeq CRISPR Analysis System is designed for multiplex analysis of on- and off-target editing, it also performs exceptionally well on batch analysis for single amplicon designs. Its analyses of on-target editing have high concordance with traditional workflows but offer a more simplified and cost-effective workflow, supporting high-throughput screening applications.

Figure 4. The rhAmpSeq CRISPR system supports a robust, complete workflow for high-throughput, batch single amplicon design and analysis for on-target editing. (A) A batch of single amplicon assays (n=96) were designed using the rhAmpSeq Design Tool or Primer3 (primers spaced >20 bp from the cut site with 130 bp design space) with default parameters, and the features/design success rates were compared. The rhAmpSeq Design Tool was more supportive than Primer3 to batch primer design. (B) Primers designed by Primer3 were synthesized as DNA-only primers, while primers designed by the rhAmpSeq Design Tool were synthesized as end-blocked, RNaseH2-cleavable rhAmp primers. DNA-only or rhAmp primers were used to amplify CRISPR-edited gDNA samples from HEK293 cells using either Q5® Hot Start High-Fidelity 2x Master Mix (NEB) or rhAmpSeq Master Mix. (C) NGS libraries from each combination of design/amplification were prepared and sequenced on an Illumina MiSeq system (2x150) and analyzed using the rhAmpSeq CRISPR Analysis Tool. Editing (% indels) concordance demonstrates that editing quantification between the two assay design strategies is equivalent.

User guides and protocols

Sample data

For more information, refer to the analysis guidelines in the User guides and protocols section.

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