CRISPR‐Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella ) patens
CRISPR‐Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella ) patens
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苔藓立碗藓（以前称为立碗藓）专利中的 CRISPR-Cas9 基因组编辑
DOI：
10.1002/cpz1.725
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Bezanilla, Magdalena
中科院分区：
文献类型：
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作者：
Wu, Shu‐Zon;Ryken, Samantha E.;Bezanilla, Magdalena
直到最近，精确的基因组编辑一直局限于少数几个生物。Cas9在特定的基因组位置产生双链DNA断裂的能力极大地扩展了许多生物和细胞类型的分子工具箱。在CRISPR-Cas9介导的基因组编辑之前，通过同源重组整合DNA的能力在植物中是独一无二的。然而，需要对同源重组事件进行选择才能获得编辑的植物，从而限制了可能的编辑类型。现在有了CRISPR-Cas9，分子操作INP。专利大大扩大了。该协议描述了一种生成各种不同基因组编辑的方法。该协议描述了一种简化的方法，以生成Cas9/sgRNA表达构建体，设计同源模板，转化植物，并快速对植物进行基因分型。2023 Wiley期刊LLC.基本协议1：构建Cas9/sgRNA瞬时表达载体备选协议1：生成单个和混合Cas9/sgRNA表达载体的快捷方式基本协议2：设计基于寡核苷酸的同源定向修复模板替代协议2：设计基于质粒的HDR模板基本协议3：通过转换CRISPR载体在Top中诱导基因组编辑。专利原生质体基本协议4：识别编辑过的植物。
Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vectorAlternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectorsBasic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) templateAlternate Protocol 2: Designing the plasmid‐based HDR templateBasic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplastsBasic Protocol 4: Identifying edited plants.