Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States.
IAME, UMR 1137, INSERM, Universités Paris Diderot et Paris Nord, Paris, 75018, France.
ACS Synth Biol. 2020 May 15;9(5):1083-1099. doi: 10.1021/acssynbio.9b00440. Epub 2020 Apr 28.
In , editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
在，由于 Cas9：gRNA 介导的 DNA 双链断裂 (DSB) 诱导的细胞死亡水平的不同，Cas9 介导的重组效率在不同的靶标之间有所差异。我们发现，使用相同的 gRNA 和修复模板，编辑效率也会随着靶标位置、启动子强度和生长条件的变化而变化。不完全编辑、脱靶活性、非靶向突变以及即使 Cas9 结合也不能切割靶标 DNA 都会影响编辑效率。这些对编辑效率的影响是 gRNA 特异性的。我们提出，Cas9：gRNA 介导的 DNA DSB 的效率差异，以及 Cas9：gRNA 复合物与其靶位点结合的可能差异，解释了 gRNA 之间观察到的编辑效率变化。我们表明，通过突变 gRNA 间隔区可以改变 DNA DSB 活性，从而改变使用相同 gRNA 的编辑行为。最后，我们讨论了不同 gRNA 的可变编辑如何限制高通量应用，并提供了克服这些限制的策略。
