How do I design a rhAmpSeq™ CRISPR Panel to evaluate on- and off-target editing using the rhAmpSeq CRISPR Analysis Tool?
For compatibility with the rhAmpSeq CRISPR Analysis Tool, we recommend submitting the targets of interest to the rhAmpSeq 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 –).
Additionally, consider the following criteria with the BED file input:
- 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 can adversely affect data analysis.
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.
Only the following organisms are supported by rhAmpSeq CRISPR Analysis Tool:
- Homo sapiens, human (hg38 & hg19)
- Mus musculus, mouse (mm10)
- Caenorhabditis elegans (Wbcel235)
- Rattus norvegicus, rat (rn6)
- Danio rerio, zebrafish (Grcz11)
- Macaca mulatta, rhesus monkey (rheMac8)
- Macaca fascicularis, crab-eating macaque (macFas5)