The target should typically have the sequence motif N21GG. The first 20 bases form the “protospacer” and they must to be incorporated as part of the guide RNA that will localize the Cas9 DNAse to the target site. These bases are required to pair with the target complementary DNA strand. The CRISPR/Cas9 mechanism can apparently utilize almost any sequence for the unspecified bases. Although some target sequences can apparently exhibit varying cleavage efficiencies, the cleavage efficiency at a specific site cannot currently be predicted based on the characteristics of the target sequence. Therefore, there may be occasions where targets need to be tested empirically.
The last 3 bases of the N21GG motif (that is, ...NGG) are the PAM (protospacer- adjacent motif). The last GG dinucleotide is required by the S. pyogenes Cas9 for efficient target recognition and interacts directly with the Cas9 protein. Deviations from this motif significantly reduce efficiency of cleavage . In the near future, Cas9 variants may be engineered that utilize different PAM motifs. Finally, some Cas9 proteins from other bacterial species can apparently have different PAM recognition motifs , so the array of possible CRISPR/Cas targets may increase as alternate Cas variants become available.
• Are there required sequence characteristics within the 20-base base-pairing region? To efficiently synthesize the guide RNA, the RNA polymerase will usually prefer certain bases in the first 1 or 2 bases of the protospacer sequence. Note that these are defined by the type of RNA polymerase used to synthesize the guide RNA, not the CRISPR/Cas9 mechanism itself. For in-vitro synthesized guide RNAs, T7 RNA polymerase will be typically employed. T7 RNA Pol “prefers” GG as the first two bases. If the guide RNA is to be expressed inside the target cell from an injected (or transfected) DNA plasmid, the vector will often contain the human U6 snRNA promoter for this purpose. This promoter is transcribed by RNA Pol III which prefers G as the first base.
Other than that, there are apparently few strict limits on the sequence content of the protospacer. However, efficiency of guide RNA loading onto Cas9 is optimized when the last 4 protospacer bases are purines and U is avoided (T in the target sequence) . Also, avoid targets with stretches of 4 or more T’s in a row as they can impair guide RNA transcription. TTTTT is the termination signal for RNA Pol III.
• Can the protospacer sequence be lengthened to improve efficiency or specificity? No. 
• Can the protospacer be shortened? Surprisingly, the protospacer can apparently be shortened by trimming off 1-3 bases from the 5’ end, with negligible loss in cleavage efficiency and reduction in off-target cleavage .
• Where in the target does Cas9 cleave the DNA? The Cas9-mediated strand cleavage sites are within the protospacer, close to the PAM motif. Cas9 normally cleaves both strands. The precise cleavage site on either strand can vary slightly  but map within the colored regions below:
Green/Magenta: Sites of cleavage by Cas9, from Jinek et al. Science 337, 816 (2012). Gray: PAM sequence.
• Identifying potential CRISPR targets in genes of interest: The PAM motif is reasonably frequent in mammalian DNA. Many or most genes will have enough targets to be amenable to some form of mutagenesis with this motif, so we suggest it as a starting point for target searching in your gene of interest. Also, a CRISPR target design tool is available: http://crispr.mit.edu.
In the near future, S. pyogenes Cas9 variants may be created that utilize different PAM motifs (but not as of early 2014). Finally, some Cas9 proteins from other bacterial species can apparently have different PAM recognition motifs , so the array of possible CRISPR/Cas targets may increase as alternate Cas variants become available.
Next post: Specificity of CRISPR/Cas9 cleavage.
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