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Using the Cas9 nickases can allow improved specificity for generating a double-strand break (DSB) in DNA. Two crRNAs are required to create a DSB using the nickases, one targeting each strand near the target location. This effectively doubles the required recognition sequence from 20 to 40 bases, decreasing the likelihood of an off-target DSB. This makes the Cas9 nickase a good option for experiments requiring a high level of specificity; however, the system can be more challenging to set up, because multiple elements must work together. To learn more about the design and use of Cas9 nickases, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Resources section).