CRISPR genome editing generates double-stranded breaks (DSBs) in genomic DNA and is a targeted method by which to achieve gene knockouts and knock-ins. Endogenous repair of DSBs results in an array of possible outcomes including insertions, deletions, and substitutions. Regions of the genome that have sufficient homology to the delivered guide RNA (gRNA) may also be cleaved, resulting in potential off-target genomic edits. Next generation sequencing (NGS) via amplicon enrichment is one method of quantifying both on- and off-target edits. Deep sequencing across the target region (on-target) and potential off-target genomic loci is essential to understand the outcome of genome editing experiments with regards to quantifying efficiency and specificity.
Amplicon sequencing is a targeted NGS method that uses PCR to create sequences of DNA called amplicons. For amplicon sequencing, IDT has developed the rhAmpSeq system, which allows precise PCR amplification, generating highly multiplexed libraries ready for amplicon sequencing on Illumina NGS platforms. The rhAmpSeq system is based on our proprietary RNase H2-dependent PCR (rhAmp™ PCR) technology that easily facilitates panel design and uses a high-throughput workflow to generate NGS-ready amplicon libraries for deep, targeted sequencing.
Here, we present the rhAmpSeq CRISPR Analysis System, an end-to-end solution enabling primer design, amplicon library generation, and NGS analysis of single or multiplex CRISPR on- and off-target editing data (Figure 1). The rhAmpSeq CRISPR Analysis Tool provides an intuitive graphical interface to analyze, explore, and generate publication-ready reports without the need for advanced bioinformatics skills. Some advantages of the rhAmpSeq CRISPR Analysis System are:
- Capture diverse allelic events with optimized primer design for single or multiplex amplification
- Perform only two PCR amplification steps in a fast, easy workflow
- Achieve cost-effective library preparation using custom panels combining on- and off-target potential editing sites into pooled amplification reactions
- Reduce the formation of primer dimers or misprimed PCR products
- Obtain an accurate, publication-ready, CRISPR NGS editing report using the fully compatible rhAmpSeq CRISPR Analysis Tool.
The first step in a CRISPR editing analysis workflow is the CRISPR editing (Figure 1). For CRISPR editing, the Alt-R CRISPR system of design tools, Cas nucleases, guide RNAs, and other relevant reagents will provide all the necessary components to edit your target sites. For more information, see The CRISPR basics handbook.
Since the rhAmpSeq CRISPR Analysis System provides a targeted sequencing method, it first requires identification of potential targets, or off-target nomination (Figure 1). On-target sequences are the sequences that are targeted for CRISPR editing, so these sequences are known, but off-target sequences must be nominated using either empirical methods (e.g., GUIDE-seq , CIRCLE-seq , Digenome-seq , DISCOVER-seq , or other empirical methods) or in silico tools (e.g., IDT CRISPR-Cas9 guide RNA design checker) that attempt to identify off-target, double-stranded breaks that may occur in the genome. There are differences between biochemical (in vitro), cell-based, or in vivo methods in terms of the resulting nominated off-target sites. At IDT, we often design our rhAmpSeq CRISPR panels to contain empirically determined off-target sites (from at least one method) in addition to the top non-overlapping in silico predicted off-target sites. The rhAmpSeq system’s high multiplexing capability enables broad coverage of empirically-defined, off-target sites as well as expanded capacity to include in silico-identified off-target sites, a strategy that has been employed by our team collaboratively  as well as recommended by regulatory agencies in pre-IND filings.
Off-target quantification: rhAmpSeq CRISPR Panel design
After the on- and off-target sites are nominated, the rhAmpSeq Design Tool designs the rhAmpSeq CRISPR Panels (Figure 1). A rhAmpSeq CRISPR Panel is a collection of rhAmpSeq CRISPR assays; each of these assays consists of one forward and one reverse rhAmp Primer designed to amplify a specific DNA region. Within a rhAmpSeq CRISPR Panel, the rhAmpSeq CRISPR Forward Pool contains all the Forward rhAmp Primers, while the rhAmpSeq CRISPR Reverse Pool contains all the Reverse rhAmp Primers. The rhAmpSeq Design Tool aims to maximize PCR target specificity at intended loci and minimize any amplification of unintended loci that might consume valuable sequencing reads. Therefore, on occasion, the design output will also include not only the main panel but also a secondary pool and possibly individual (singleton) primer sets requiring independent amplification. See the table below for an example of a “promiscuous” editing target that required a more complex panel design. The aim was to design panels for one on-target site and 199 potential off-target locations. Even though secondary pools and a singleton assay were necessary for this optimized design, only six total PCRs were required for rhAmpSeq sequencing, as opposed to the 400 that would have been necessary for traditional amplicon sequencing.
|Gene||Primary pool||Secondary pool||Singletons||Total libraries/samples||Total designs||On-target (Chr/Start)||Total PCRs rhAmpSeq||Total PCR traditional amplicon sequencing|
Several factors may require multiple panel designs:
- The PCR targets may be too close together. Targets that are less than 500 bp apart preclude usage of two unique primer pairs in a single pool. Typically, this results in an adjacent target needing to be covered by either a secondary pool or as a singleton assay, if the assays cannot be merged.
- The algorithm may not be able to select a primer pair that is both specific enough to cover the PCR target of interest and unique enough to allow pooling (multiplexing) without creating primer dimers.
- The optimal primer pair for a given target may amplify a repetitive region. In this case, the optimal primer pair must remain a singleton primer pair to avoid impacting the performance of the primary pool.
- An assay with potential performance issues is less likely to be pooled due to the risk of lowering overall panel performance. Examples of such potential performance issues include the following: (1) a primer overlaps common single nucleotide polymorphisms (SNPs) leading to potential Cq delay, (2) primer or amplicon GC content significantly differs from the panel’s average GC content, (3) primers have non-ideal Tm or secondary structure, (4) amplicons contain homopolymers, or (5) the locus and design constraints do not allow for design of a primer pair that is expected to amplify uniformly with the assays in the primary pool.
BED file submission for efficient panel design
For compatibility with the rhAmpSeq CRISPR Analysis Tool (CRISPAltRations), IDT recommends submitting the targets of interest to the rhAmpSeq Primer Design Tool as a 6-column BED file that includes the following information: chromosome, start, stop, desired name, mismatch # (or a 0), and strand (+ or -).
- The BED file needs to be devoid of headers.
- The input to the tool should be the coordinates of your on- and off-target gRNA binding loci (i.e., usually 20–24 bases in length)
- The guides should include “strand” information. This is needed for the rhAmpSeq CRISPR Analysis Tool to report results accurately. Failing to provide strandedness will result in use of an incorrect, non-optimal editing window in the analysis software.
- Guide locations should NOT include the PAM sequence. Remove the PAM sequence from the target location in the BED file. Failure to remove all the PAM information will also adversely affect data analysis.
Note that the rhAmpSeq CRISPR Analysis Tool has the following run restrictions:
- Multiplexed amplification pools are limited to 500 target sites per pool
Input FASTQ files should be <1 GB in file size.
Target validation and publication
The PCR products from the rhAmpSeq PCR reactions are run on an Illumina NGS instrument and sequenced. The rhAmpSeq CRISPR Analysis Tool analyzes the raw sequencing data and provides validation of editing of the targeted site along with a publication-ready report of off-target edits. Together, our data indicate that the rhAmpSeq CRISPR Analysis Tool outperforms other tools for characterization of CRISPR editing.