Choosing a Target Sequence for CRISPR/Cas9 Gene Editing
CRISPR/Cas9 gene targeting requires a custom single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a Cas9 nuclease-recruiting sequence (tracrRNA). The crRNA region (shown in red below) is a 20-nucleotide sequence that is homologous to a region in your gene of interest and will direct Cas9 nuclease activity.
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Use the following guidelines to select a genomic DNA region that corresponds to the crRNA sequence of the sgRNA:
- The 3' end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3'). The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA) and Cas9 nuclease will cleave approximately 3 bases upstream of the PAM.
- The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.
- The target sequence can be on either DNA strand.
There are several online tools (e.g., CRISPR Design or CHOPCHOP) that detect PAM sequences and list possible crRNA sequences within a specific DNA region. These algorithms also predict off-target effects elsewhere in the genome, allowing you to choose the most specific crRNA for your research application. Please visit our Online Tools for Guide RNA Design page to learn more.
Tips for designing sgRNAs
Through our own experience, we have identified additional tips for designing sgRNAs. We have found that the best sgRNAs for several tested genes have a G at position 1 and an A or T at position 17.
Synthesize your sgRNA with the Guide-it sgRNA In Vitro Transcription Kit. With this kit, PCR is used to generate a template DNA containing the sgRNA-encoding sequence (constituted by your crRNA and a tracRNA) under the control of a T7 promoter. Then, in vitro transcription using the PCR product template is used to produce sgRNAs that can be purified for efficiency testing and/or cell transduction.
Using the most effective sgRNA
Even if you pick a target sequence that fulfills all of the described requirements, sgRNA specificity and activity is unpredictable. Therefore, it is often recommended that multiple, different sgRNAs are designed to target a gene of interest. Our Guide-it sgRNA Screening Kit provides a simple and robust in vitro method for assessing sgRNA efficiency before cell transduction, allowing you to identify the most effective CRISPR.