The rhAmp™ primer pairs in rhAmpSeq CRISPR Panels mitigate primer dimers and maximize multiplexing to provide you with high-quality amplicon libraries. Analyze more samples faster with the confidence of uniform coverage and the time savings of efficient library prep. The rhAmpSeq Design Tool makes it easy to input your targets, and our speedy delivery means you can validate your CRISPR results sooner. With our rhAmpSeq CRISPR Panels you have several advantages:
rhAmpSeq CRISPR Panels are highly multiplexed primer sets for targeted sequencing. These panels have been expressly designed for and successfully tested with a wide range of species, including human (hg19, hg38), non-human primate (rhesus monkey), mouse, rat, zebrafish, and nematode (C. elegans).
rhAmpSeq CRISPR Panels are Custom rhAmpSeq Panels specifically designed to validate CRISPR edits. These panels are ideal for confirming on- and off-target CRISPR gene editing experiments.
Our rhAmpSeq Design Tool lets you quickly and easily design custom rhAmpSeq CRISPR Panels specific to your application and targets of interest (Figure 1). If you would like additional assistance, our expert bioinformatics team can help tailor a design for your custom project (firstname.lastname@example.org).
Once your design is complete, we provide a detailed summary report of the custom panel results for you to review and, when necessary, iterate your design before you order your custom rhAmpSeq CRISPR Panel. Also available for download are the associated assay BED files and assay IDs to order any sub-panel from the set of assays that were a part of your custom design.
Custom assay design involves two key steps that provide improved performance over other amplicon sequencing systems:
Any assay that is successfully designed but not able to be pooled into the primary panel will be placed in secondary panels. These secondary panels have a designated prefix (such as P2 or SC) in the resulting panel name that correlates to user input during the design submission. Refer to the rhAmpSeq Design Tool User Guide for more information.
Custom rhAmpSeq CRISPR Panels are available in three convenient scales to fit your experimental needs: 0.4 nmol, 4 nmol, and 8 nmol. As shown in Table 1, we recommend adjusting rhAmp primer concentrations in the first amplification reaction (Targeted rhAmp PCR 1) depending on your panel size (plexity). Table 1 shows the approximate number of reactions for each scale based on panel size.
Table 1. Number of rhAmp primer reactions based on scale and panel size.
|Panel size||rhAmp primer concentration in 10X panel*||Number of reactions per rhAmp primer order||Example|
|Panel size||Number of reactions|
|0.4 nmol primer (x)||4 nmol primer (x)||8 nmol primer (x)|
|≥500-plex||50 µM total primers||x nmol of primer/(0.1 nmol total/panel size)||1000||4000||40,000||80,000|
|101-499–plex||100 nM each primer||x nmol of primer/0.0002 nmol per rxn||400||2000||20,000||40,000|
|≤100-plex||250 nM each primer||x nmol of primer/0.0005 nmol per rxn||100||800||8000||16,000|
* Important: When creating rhAmpSeq primer pools, do not to combine forward and reverse primers for long-term storage. The primer concentrations in the 10X panel stocks assume there will be a separate forward pool of primers and a separate reverse pool of primers. Refer to the protocol in the Resources section for details.
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 2). 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 2. 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 1-29) is reported using an untreated control for background subtraction. Background editing was <0.2% for all assays in this panel.
For more information, refer to the analysis guidelines in the User guides and protocols section.