用于柑橘胚性细胞系基因组编辑的CRISPR-Cas12a核糖核蛋白复合物的递送
Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.
作者信息
Fang Hong, Culver James N, Niedz Randall P, Qi Yiping
机构信息
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
出版信息
Methods Mol Biol. 2023;2653:153-171. doi: 10.1007/978-1-0716-3131-7_10.
Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.
成簇规律间隔短回文重复序列（CRISPR）技术是一种强大的基因组编辑工具。最近开发的CRISPR-Cas12a系统相对于CRISPR-Cas9具有多个优势，使其成为植物基因组编辑和作物改良的理想选择。虽然基于质粒递送的传统转化方法存在与转基因整合和脱靶效应相关的问题，但以核糖核蛋白（RNP）形式递送的CRISPR-Cas12a可以有效缓解这些潜在问题。在此，我们展示了一种在柑橘原生质体中使用RNP递送进行LbCas12a介导的基因组编辑的详细方案。该方案为RNP组分制备、RNP复合物组装与递送以及编辑效率评估提供了全面的指导方针。
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