圖片來源：www.phys.org

為了闡明特殊基因錯(cuò)誤如何引發(fā)疾病，科學(xué)家們需要在細(xì)胞中進(jìn)行實(shí)驗(yàn)來研究具體突變對細(xì)胞的影響，如今來自洛克菲勒大學(xué)（Rockefeller University）和紐約干細(xì)胞研究所等機(jī)構(gòu)的研究人員通過研究，利用基于CRISPR的基因編輯技術(shù)成功在細(xì)胞中重現(xiàn)了疾病發(fā)生的過程，相關(guān)研究刊登于國際著名雜志Nature上。

研究者M(jìn)arc Tessier-Lavigne說道，這種新型技術(shù)可以幫助科學(xué)家們直接精確地將引發(fā)疾病發(fā)生的基因植入細(xì)胞中，從而獲取細(xì)胞模型來進(jìn)行更為深入的研究，這就為后期開發(fā)一系列人類疾病的新型療法提供了新的希望，比如治療阿爾茲海默氏癥等。

過去很多年里，科學(xué)家們設(shè)計(jì)了很多種方法來模擬在實(shí)驗(yàn)室培養(yǎng)的細(xì)胞中模擬疾病的發(fā)生過程，當(dāng)科學(xué)家們盡力想讓細(xì)胞轉(zhuǎn)變成為特殊人類疾病模型時(shí)，他們就通過切割基因組中的DNA并且換上替代品來進(jìn)行研究。隨著CRISPR-Cas9系統(tǒng)的發(fā)現(xiàn)，科學(xué)家們開始利用基于該系統(tǒng)的基因編輯技術(shù)來開發(fā)出患病的細(xì)胞模型。

文章中研究人員Dominik Paquet及其同事首次嘗試?yán)肅RISPR-Cas9技術(shù)在細(xì)胞中插入兩種遺傳突變，而這兩種遺傳突變和引發(fā)阿爾茲海默氏癥疾病的β淀粉樣蛋白的產(chǎn)生直接相關(guān)，隨后研究者發(fā)現(xiàn)，這種方法的成功率較低，僅有一小部分細(xì)胞會(huì)攜帶上理想的基因突變。主要的問題就是CRISPR-Cas9可以持續(xù)切割細(xì)胞的DNA，而細(xì)胞的自身修復(fù)細(xì)胞會(huì)不斷修復(fù)每一個(gè)切割處直到細(xì)胞產(chǎn)生一種可以抑制切割的錯(cuò)誤，而這種錯(cuò)誤一旦產(chǎn)生就會(huì)在細(xì)胞中不斷產(chǎn)生很多新型未知的問題。

隨后科學(xué)家們評估了另外一種方法，即引入大塊的突變來抑制后期的切割現(xiàn)象，通過在CRISPR-Cas9靶向檢測的DNA不同部位中引入大塊突變后，研究者發(fā)現(xiàn)這可以明顯減少意外錯(cuò)誤產(chǎn)生的數(shù)量。當(dāng)研究人員利用CRISPR-Cas9引入阿爾茲海默氏癥的任意一種遺傳突變后，他們仔細(xì)觀察遺傳序列后發(fā)現(xiàn)了一種特殊的模式，也就是說在CRISPR-Cas9切割位點(diǎn)和研究者引入受體細(xì)胞的突變之間存在一段序列上的距離。

隨后研究者Kwart說道，序列距離較短會(huì)產(chǎn)生出更易于包含兩種突變的細(xì)胞，而隨著距離增加，編輯的成功率就會(huì)降低，而一種突變的比率和其原始基因版本的峰值之間的距離就會(huì)開始拉大；更為重要的是研究者發(fā)現(xiàn)了特殊的距離關(guān)系，這樣他們就有可能制造出大量的雜合細(xì)胞；而利用上述技術(shù)研究人員就可以對干細(xì)胞的基因組進(jìn)行編輯使細(xì)胞包含兩種阿爾茲海默氏癥基因中的任意一種，隨后誘導(dǎo)這些干細(xì)胞轉(zhuǎn)化成為神經(jīng)元細(xì)胞并且產(chǎn)生大量β淀粉樣蛋白，從而模擬阿爾茲海默氏癥的疾病表現(xiàn)。

此前并沒有簡單的方法來控制確定是否通過CRISPR-Cas9技術(shù)編輯就可以產(chǎn)生和特殊疾病表現(xiàn)相關(guān)的雜合突變，而本文研究中，研究者通過對上述距離關(guān)系特性的分析就成功實(shí)現(xiàn)了利用基于CRISPR的技術(shù)在細(xì)胞中重現(xiàn)疾病癥狀的目的。（世聯(lián)博研(Bioexcellence)世聯(lián)博研Bioexcellence）

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doi:10.1038/nature17664
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Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9

Dominik Paquet, Dylan Kwart, Antonia Chen, Andrew Sproul, Samson Jacob, Shaun Teo, Kimberly Moore Olsen, Andrew Gregg, Scott Noggle & Marc Tessier-Lavigne

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells1, 2. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels)2. DSBs may also be repaired by homology-directed repair (HDR)1, 2 using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations3. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient3, 4, 5, 6, 7, 8 and can be corrupted by additional indels9, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ‘CORRECT’ for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation’s incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer’s disease-causing mutations in amyloid precursor protein (APPSwe)10 and presenilin 1 (PSEN1M146V)11 and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.