![]() ![]() Definitely should be less than 100bp away, ideally less than 10bp away.įor small (100bp insertions or deletions), we typically use plasmid donor, with two homology arms on each side flanking your desired insertion or mutation. And it is best if the cut site is as close to the junction of the homology arm as possible. A good reference is this Cell paper: (13)01015-5Īs for target selection, we usually pick 3-6 guides around the region to find the most efficient guide (most guides would work, but they sometimes have different efficiency). Alternatively, a new system described recently is the double nickase system, with PX335 vector, you need to find two guides for each cleavage site, but this system probably have better specificity. Generally if off-target (non-specific) cleavage is not a very big concern, you could go ahead with PX330 plasmid, clone in your target guides, test run them and then select the best guide to co-transfect in HR donor. Should I add the PAM sequence to the oligo when cloning my target spacers into the PX330 vector backbone?Ī few notes below are considerations for designing HR donor.For genome editing, the Zhang lab has published this Nature Protocol article. Many protocols have been published with thorough step-wise instructions on designing and using Cas9-based genome engineering tools to perform different types of genome targeting. ![]() There are a few references for the double nickase system, including one recently from the Zhang group. At the same time, the off-target effects are reduced because the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does. The concept of the double nickase system is that you can express two different chimeric gRNAs with the Cas9 nickase which will together introduce cleavage of the target site with efficiency similar to using a single chimeric gRNA. For example, if you want to use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, et. 'Double nickase' is a new system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design but with better accuracy (in other words, lower off-target effect). When assessing which nickase type to use for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency but has been shown to have off-target effects.
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