将精确的点突变敲入蛋白质编码基因一直是簇状规则间隔的回文重复序列（CRISPR）/ Cas9的最早也是最重要的应用之一。 执行这种精确的基因编辑的能力对于询问特定蛋白质残基的功能以及创建由蛋白质氨基酸变化引起的人类疾病的模型至关重要。 同源蛋白残基可以在模型动物物种中突变，并且可以研究这些突变的后果，从而使人们对所讨论的疾病有了更好的了解。 点突变敲入策略的设计是由多个计算工具辅助的手动步骤的组合，这导致了一个耗时的过程并阻止了一个快速而综合的解决方案。 因此，我们设计了CRISPR敲入设计器，可以在提供突变，指导RNA以及必要的标识符或序列信息后，快速自动地设计点突变敲入DNA寡核苷酸。 该工具支持大多数实验建立的CRISPR类型，并为所得寡核苷酸提供多种选择，以满足大多数用户的需求。 我们还提供基于等位基因的特定PCR和基于限制酶的基因分型策略，作为程序输出的一部分。 访问网站： https://crisprtools.shinyapps.io/knockinDesigner/ CRISPR敲入设计器会适应任何目标密码子的基因组背景，并尝试设计一个密码子跨越两个外显子时的敲入策略，这种情况我们已在几种模型物种的整个基因组中进行了探讨。 CRISPR敲入设计器输出也可以与某些较新的Prime Editing设计工具配合使用，以使用此先进技术促进特定突变序列的引入。 Knock-in of precise point mutations into protein-coding genes has been one of the earliest and most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. The ability to perform such precise gene editing is crucial to interrogate the function of specific protein residues and to create models of human diseases caused by protein amino acid changes. The homologous protein residues can be mutated in model animal species, and the consequences of these mutations can be studied, leading to a better understanding of the disease in question. Design of point mutation knock-in strategies has been a combination of manual steps assisted by several computational tools resulting in a time-consuming process and preventing a single rapid and integrated solution. We have therefore designed CRISPR Knock-in Designer, which can perform rapid and automatic design of point mutation knock-in DNA oligonucleotides upon provision of the mutation, a guide RNA, and essential identifier or sequence information. The tool supports most experimentally established CRISPR types and has multiple options for the resulting oligonucleotides to satisfy the needs of most users. We also provide allele-specific PCR-based and restriction enzyme-based genotyping strategies as part of the program output. CRISPR Knock-in Designer adjusts to the genomic context of any target codon and tries to design knock-in strategies when a codon straddles two exons, a situation we explored in whole genomes of several model species. CRISPR Knock-in Designer output can also be adapted for use with some of the newer Prime Editing design tools to facilitate the introduction of a specific mutation sequence using this advanced technology.
