从用于基因组编辑的CRISPR/gRNA引物的设计到验证
From design to validation of CRISPR/gRNA primers towards genome editing.
作者信息
Jamee Mohd Rizwan, Ansari Zubaida, Qureshi Mohammad Irfan
机构信息
Department of Biotechnology, Jamia Millia Islamia, New Delhi - 110025, India.
Centre for Interdisciplinary Research in Basic Science, Jamia Milia Islamia, New Delhi - 110025, India.
出版信息
Bioinformation. 2022 May 31;18(5):471-477. doi: 10.6026/97320630018471. eCollection 2022.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. There are many online platforms/software for the design of gRNAs and related primers. However, there are concerns in design regarding off-site deletions besides knocking out sequences in the target genes. Nonetheless, a well known robust platform for CRISPR/gRNA primers design is CRISPRdirect. We demonstrate the use of this tool in the design of CRISPR/gRNA primers for soluble starch synthases (SSS) II-1, 2, and 3 genes in the Oryza sativa genome followed by the PCR-mediated amplification of SSS genes with corresponding confirmation towards genome editing having improved phenotype features.
CRISPR（成簇规律间隔短回文重复序列）/Cas9（CRISPR相关系统）用于精确且高效地编辑特定基因组序列。有许多在线平台/软件可用于设计向导RNA（gRNA）和相关引物。然而，在设计过程中，除了敲除靶基因中的序列外，还存在脱靶缺失的问题。尽管如此，一个著名的用于CRISPR/gRNA引物设计的强大平台是CRISPRdirect。我们展示了该工具在设计水稻基因组中可溶性淀粉合酶（SSS）II-1、II-2和II-3基因的CRISPR/gRNA引物中的应用，随后通过PCR介导对SSS基因进行扩增，并对具有改善表型特征的基因组编辑进行相应确认。
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