Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
Nat Plants. 2021 Jan;7(1):25-33. doi: 10.1038/s41477-020-00827-4. Epub 2021 Jan 4.
The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G), limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. ) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.
CRISPR-Cas9、-Cas12a 和 -Cas12b 基因组编辑系统的快速发展极大地推动了基础和转化植物研究。这些 Cas 核酸酶对 DNA 的靶向作用受到其偏好的原间隔序列邻近基序 (PAM) 的限制。最受欢迎的酿脓链球菌 Cas9 (SpCas9) 的 PAM 要求是 NGG (N = A、T、C、G)，限制了其靶向 GC 丰富区域的范围。在这里，我们使用工程化的 SpRY Cas9 变体在水稻（单子叶植物）和落叶松（针叶树）中在放松的 PAM 位点进行基因组编辑。通过 SpRY 在落叶松原生质体和水稻转基因系中非同源末端连接 (NHEJ) 中在放松的 PAM 位点可以轻松实现高效靶向突变。此外，通过 SpRY 开发了基于胞嘧啶碱基编辑器，并通过在水稻中定向进化新的除草剂抗性 OsALS 等位基因进行了证明。同样，基于 ABE8e（参考文献）开发了高度活跃的 SpRY 腺嘌呤碱基编辑器，并且 SpRY-ABE8e 能够靶向水稻植物中的放松的 PAM 位点，实现高达 79%的编辑效率和高产物纯度。因此，SpRY 工具包通过以无 PAM 的方式进行 DNA 编辑，打破了植物基因组工程中的 PAM 限制障碍。由于 SpRY 介导的转移 DNA 自我编辑，还提供了新的单指导 RNA (sgRNA) 引起的次级脱靶效应的证据，这需要在实施 SpRY 基因组编辑工具包时设计高度特异性 sgRNA 的更复杂方案。
