高通量分析 Cas9 变体特异性。
Highly Parallel Profiling of Cas9 Variant Specificity.
机构信息
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA.
出版信息
Mol Cell. 2020 May 21;78(4):794-800.e8. doi: 10.1016/j.molcel.2020.02.023. Epub 2020 Mar 17.
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.
确定可编程核酸酶的脱靶切割谱是任何基因组编辑实验的一个重要考虑因素，已经报道了许多可以提高特异性的 Cas9 变体。我们在这里描述了基于标签化的标签整合位点测序（TTISS），这是一种高效、可扩展的分析双链断裂（DSB）的方法，我们将其应用于 59 个靶标中的 8 种 Cas9 变体。此外，我们还生成了数千种其他 Cas9 变体，并筛选出具有增强特异性和活性的变体，确定了 LZ3 Cas9，这是一种具有独特+1 插入谱的高特异性变体。这项全面的比较揭示了 Cas9 活性和特异性之间的一般权衡，并提供了有关+1 插入产生频率的信息，这对纠正移码突变具有影响。
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