The CRISPR Design Tool enables targeting any region of a genome, even in non-standard species
The use of CRISPR-Cas9 systems for genome engineering has made gene editing a routine application for many researchers. One challenging aspect of CRISPR-Cas9 gene editing is the design of guide RNAs. Without bioinformatic help, guide RNA design can be a complex and daunting task. Guide RNAs predesigned to knockout gene expression are very helpful to researchers if that is their goal. For performing protein knockout of human, mouse or rat genes, Dharmacon has predesigned crRNA and sgRNA reagents already available!
However, if homology directed repair is the goal, other regions of a gene could be the area of interest. Additionally, targeting a non-coding region such as an intron, promoter region, UTR or intergenic region could be the goal. For all of these reasons, tools to assist the design of custom guide RNAs are needed.
To help with the design of guide RNAs for genome engineering experiments, the Dharmacon CRISPR Design Tool enables scientists to design their own guide RNA sequences for the Streptococcus pyogenes CRISPR-Cas9 system, and to order these custom reagents as either synthetic crRNAs or lentiviral sgRNA.
The CRISPR Design Tool uses design rules defined in the literature and internal design expertise to define default targeting and specificity settings for protein, microRNA or non-coding RNA gene loci. However, if researchers want to design their own CRISPR RNAs for unique applications, the Dharmacon CRISPR Design Tool offers several options.
The “Input a gene ID or gene symbol” option allows for design of guide RNAs to not only human, mouse and rat genes, but also Chinese hamster and zebrafish genes! The CRISPR Design Tool automatically inputs the recommended default parameters based on whether the design is for protein coding, microRNA or non-coding genes. The default parameters include a specificity check that excludes any target sites that have = 2 mismatch alignments elsewhere in the genome and are next to a PAM. The CRISPR Design Tool then outputs a list of guide RNA sequences in the order of gene position.
The output for each guide RNA sequence includes information for target sequence, protospacer-adjacent motif (PAM), variants targeted, sequence location as well as a link to the UCSC Genome Browser. The information allows researchers to closely examine the genes and genomic locations targeted to rationally select the guide RNA sequence most appropriate for their particular experiment.
In addition, the CRISPR Design Tool includes Advanced Options where the researcher can customize settings for design including PAM sequence, including a specificity check and transcripts targeted and GC percentage among others.
The “Target a DNA sequence” option allows scientists to input a stretch of target DNA sequence up to 10,000 bases long and generate a list of potential guide RNAs for gene editing experiments. This option is very useful when a researcher has identified a novel gene sequence or a particular stretch of DNA sequence of interest.
Finally, the “Input my own guide RNA sequence” option allows a researcher to order a synthetic crRNA or lentiviral sgRNA using input from a publication or their own guide sequence.
All of these options are explained in greater detail in the Dharmacon CRISPR Design Tool User Guide.
Optimized tools for high-confidence genome engineering
Predesigned or custom synthesized for rapid knockout studies across many genes
Lentiviral plasmid or high-titer particles for difficult-to-transfect cells or pooled screening applications