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2021/06/22
【TriLink】研究聚焦:新鹼基編輯策略有望治療鐮刀型紅血球疾病
鹼基編輯器(base editors)是由不活化的CRISPR-Cas 和脫氨酶功能區域(deaminase domain)所組成,是以CRISPR-Cas 為中心的一種基因編輯工具,作用方式是將一種鹼基對轉換為另一種鹼基對而不會導致雙股DNA 斷裂。
為了讓鹼基編輯器識別其目標,在目標基因序列上必須存在一個三核甘酸序列-Protospacer Adjacent Motif (PAM)並且此序列需靠近切割位點,這樣的需求將鹼基編輯範圍限制在PAM 序列附近。
為了增加鹼基編輯器對可編輯目標基因的廣度,Chu等人設計了一系列的嵌入式脫氨酶鹼基編輯器 (deaminase-inlaid base editors, IBEs),Cas9內包含了一個脫氨酶功能區域(deaminase domain),研究顯示其中幾個IBE可以在規範的編輯區域之外編輯目標基因,並進一步證明了 IBE 具有精確編輯患者來源造血幹細胞中鐮刀型紅血球疾病(sickle cell disease, SCD)突變的能力。
設計良好的嵌入式脫氨酶鹼基編輯器(IBE)
可能可以規避因PAM
造成的編輯範圍限制
近年來,許多研究嘗試改善基因編輯的範圍並增加鹼基編輯器的目標範圍。其中一種較多人進行的嘗試是增加編輯窗口的寬度,但這通常會導致相鄰鹼基對發生不必要的轉換,突出了對替代方法的需求。
為了解決這個問題,Chu等人提出一種假設,認為將脫氨酶功能區域(deaminase domain)插入Cas9可以改變Cas9活性位點的位置並允許在規範範圍之外進行基因編輯。為了驗證這一理論,他們根據預先定義的標準(包括結構靈活性和與DNA目標的接近度)確定了化膿性鏈球菌Cas9中的各個位點,然後在這些位點插入TadA脫氨酶,然後重新定位Cas9的活性位點。
對30個初始設計IBE的評估顯示,其中26個在HEK293T細胞中的定義目標序列內檢測到A到G轉換。從其中,選擇了8個IBE進行進一步的表徵確認,發現其中幾個IBE具有不同程度的編輯窗口移動,這是一個重要的發現,因為PAM通常距離切割位點僅3到4個核苷酸。
嵌入式脫氨酶鹼基編輯器(IBE)
有希望成為鐮刀型紅血球疾病(SCD)
的治療策略
在初步研究成功的基礎上,Chu等人接著研究IBE在人類疾病等位基因上編輯的適用性,而現有的鹼基編輯器在很大程度上無法訪問該等位基因。他們選擇了嚴重的遺傳性血液病-鐮刀型紅血球疾病(SCD),這是由人類血紅蛋白β次單元(hemoglobin beta subunit, HBB)的編碼基因中的單一突變引起的(GAG轉變成 GTG)。這種突變會產生鐮刀狀紅血球細胞,與健康細胞相比,它們的壽命較短,並且還會在小血管中積聚和阻塞血流。
研究人員使用TriLink的CleanCap
®
AG對 IBE mRNA 進行共轉錄加帽,再將已加帽mRNA 送入患者來源的造血幹細胞,所選的IBE顯示出可將致病性鐮狀細胞血紅蛋白等位基因轉化為自然發生的變體。將這種方法開發成自體幹細胞移植療法有望為鐮刀型紅血球疾病(SCD)治療帶來顯著的臨床益處。
原始文章請見>>
A Novel Base-Editing Strategy Promises to Treat Sickle Cell Disease
Article Reference
:Chu SH, Packer M, Rees H, et al. Rationally designed base editors for precise editing of the sickle cell disease mutation.
The CRISPR Journal
. 2021; 4(2):169-177
.
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