BIoVector? AAVS1 sgRNA Cas9 plasmid is a specialized tool used for CRISPR-Cas9 genome editing that simplifies the process of integrating a gene into a specific, safe location in the human genome. It combines the three core components of the CRISPR system—the Cas9 nuclease, the single guide RNA (sgRNA), and the plasmid backbone—into a single vector.

What makes this plasmid unique is that its sgRNA is specifically designed to target the AAVS1 locus. The AAVS1 (Adeno-Associated Virus Integration Site 1) locus, located on human chromosome 19, is known as a "genomic safe harbor". This means that a gene inserted here is highly likely to be expressed stably and at high levels without causing harm to the cell or disrupting the function of other genes.

Components and Function

The plasmid contains several key elements:

• Cas9 gene: This codes for the Cas9 endonuclease, the "molecular scissors" that cuts DNA.

• AAVS1 sgRNA: This is a short RNA molecule that acts as a guide. It directs the Cas9 protein to the specific AAVS1 locus on chromosome 19.

• Plasmid backbone: This is the circular DNA molecule that carries all the components and allows for replication within bacteria.

When this "all-in-one" plasmid is introduced into human cells, the sgRNA guides the Cas9 protein to the AAVS1 site, where it creates a double-strand break. Researchers can then introduce a separate donor plasmid containing their gene of interest. The cell's natural DNA repair mechanism, called homology-directed repair (HDR), will use the donor plasmid as a template to insert the new gene precisely into the AAVS1 locus.

Benefits and Applications

Using an AAVS1 sgRNA Cas9 plasmid offers several major advantages over traditional methods of gene integration:

• Stable and Predictable Expression: Integration into a safe harbor site ensures that the inserted gene remains active and is not silenced over time, a common issue with random integration.

• Reduced Off-Target Effects: By targeting a known, well-characterized site, the risk of disrupting a critical gene or causing an adverse effect is minimized.

• Simplified Cell Line Generation: It streamlines the process of creating stable, isogenic (genetically uniform) cell lines for research, such as creating disease models or reporter cell lines.

• Precise Knock-in: This system is ideal for precise gene knock-in, a process where a new gene is inserted into a specific location in the genome.

This type of plasmid is widely used in applications like creating cell-based disease models, developing transgenic cell lines, and performing functional genomics screens.

AAVS1 sgRNA Cas9 質(zhì)粒 是一種專門用于靶向腺相關(guān)病毒整合位點(diǎn)1的CRISPR-Cas9 基因編輯的工具，它簡(jiǎn)化了將特定基因整合到人類基因組中一個(gè)安全且穩(wěn)定的位置的過程。它將 CRISPR 系統(tǒng)的三個(gè)核心組件——Cas9 核酸酶、單向?qū)?RNA（sgRNA）和質(zhì)粒骨架——組合在一個(gè)載體中。

它的獨(dú)特之處

這種質(zhì)粒的獨(dú)特之處在于，它的 sgRNA 專門設(shè)計(jì)用于靶向 AAVS1 基因座。AAVS1（腺相關(guān)病毒整合位點(diǎn) 1）基因座位于人類 19 號(hào)染色體上，它被稱為一個(gè) “基因組安全港”。這意味著，如果一個(gè)基因被插入到這個(gè)位置，它極有可能穩(wěn)定且高水平地表達(dá)，同時(shí)不會(huì)對(duì)細(xì)胞造成傷害或擾亂其他基因的功能。

質(zhì)粒的組成與功能

該質(zhì)粒包含幾個(gè)關(guān)鍵元件：

• Cas9 基因： 編碼 Cas9 核酸內(nèi)切酶，即 DNA 的“分子剪刀”，負(fù)責(zé)切割 DNA。

• AAVS1 sgRNA： 這是一種短的 RNA 分子，充當(dāng)向?qū)?。它?Cas9 蛋白引導(dǎo)至 19 號(hào)染色體上特定的 AAVS1 基因座。

• 質(zhì)粒骨架： 這是一個(gè)環(huán)狀 DNA 分子，攜帶所有元件，并允許在細(xì)菌中復(fù)制。

當(dāng)這種“多合一”質(zhì)粒被導(dǎo)入人類細(xì)胞后，sgRNA 會(huì)引導(dǎo) Cas9 蛋白到達(dá) AAVS1 位點(diǎn)，并在那里創(chuàng)建一個(gè)雙鏈斷裂。隨后，研究人員可以導(dǎo)入一個(gè)單獨(dú)的供體質(zhì)粒，其中包含他們感興趣的基因。細(xì)胞自身的 DNA 修復(fù)機(jī)制，稱為同源定向修復(fù)（HDR），會(huì)利用供體質(zhì)粒作為模板，將新基因精確地插入到 AAVS1 基因座。

優(yōu)勢(shì)與應(yīng)用

使用 AAVS1 sgRNA Cas9 質(zhì)粒相比傳統(tǒng)基因整合方法有幾個(gè)主要優(yōu)勢(shì)：

• 穩(wěn)定且可預(yù)測(cè)的表達(dá)： 整合到安全港位點(diǎn)可確保插入的基因保持活性，并且不會(huì)隨著時(shí)間的推移而沉默（這是隨機(jī)整合中常見的問題）。

• 減少脫靶效應(yīng)： 通過靶向一個(gè)已知的、特征明確的位點(diǎn)，可以最大限度地降低破壞關(guān)鍵基因或引起不良反應(yīng)的風(fēng)險(xiǎn)。

• 簡(jiǎn)化細(xì)胞系構(gòu)建： 它簡(jiǎn)化了創(chuàng)建穩(wěn)定、同源（基因一致）細(xì)胞系的過程，可用于疾病模型或報(bào)告基因細(xì)胞系的研究。

• 精確敲入： 該系統(tǒng)非常適合進(jìn)行精確的基因敲入，即將新基因插入到基因組中的特定位置。

這種質(zhì)粒廣泛應(yīng)用于創(chuàng)建基于細(xì)胞的疾病模型、開發(fā)轉(zhuǎn)基因細(xì)胞系以及進(jìn)行功能基因組學(xué)篩選等領(lǐng)域。

BioVector NTCC質(zhì)粒載體菌種細(xì)胞蛋白抗體基因保藏中心

BioVector NTCC Inc.

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