將精確的點(diǎn)突變敲入蛋白質(zhì)編碼基因一直是簇狀規(guī)則間隔的回文重復(fù)序列（CRISPR）/ Cas9的最早也是最重要的應(yīng)用之一。 執(zhí)行這種精確的基因編輯的能力對(duì)于詢問特定蛋白質(zhì)殘基的功能以及創(chuàng)建由蛋白質(zhì)氨基酸變化引起的人類疾病的模型至關(guān)重要。 同源蛋白殘基可以在模型動(dòng)物物種中突變，并且可以研究這些突變的后果，從而使人們對(duì)所討論的疾病有了更好的了解。 點(diǎn)突變敲入策略的設(shè)計(jì)是由多個(gè)計(jì)算工具輔助的手動(dòng)步驟的組合，這導(dǎo)致了一個(gè)耗時(shí)的過程并阻止了一個(gè)快速而綜合的解決方案。 

因此，我們?cè)O(shè)計(jì)了CRISPR敲入設(shè)計(jì)器，可以在提供突變，指導(dǎo)RNA以及必要的標(biāo)識(shí)符或序列信息后，快速自動(dòng)地設(shè)計(jì)點(diǎn)突變敲入DNA寡核苷酸。 該工具支持大多數(shù)實(shí)驗(yàn)建立的CRISPR類型，并為所得寡核苷酸提供多種選擇，以滿足大多數(shù)用戶的需求。 我們還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，作為程序輸出的一部分。

訪問網(wǎng)站：

https://crisprtools./knockinDesigner/ 

CRISPR敲入設(shè)計(jì)器會(huì)適應(yīng)任何目標(biāo)密碼子的基因組背景，并嘗試設(shè)計(jì)一個(gè)密碼子跨越兩個(gè)外顯子時(shí)的敲入策略，這種情況我們已在幾種模型物種的整個(gè)基因組中進(jìn)行了探討。 CRISPR敲入設(shè)計(jì)器輸出也可以與某些較新的Prime Editing設(shè)計(jì)工具配合使用，以使用此先進(jìn)技術(shù)促進(jìn)特定突變序列的引入。

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.

溫馨提示：