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目前已开发了两种主要的碱基编辑器:
CBE和ABE可有效介导四种碱基转换突变(C→T,A→G,T→C,G→A),这类突变约占当前已注释的人类病原体变异的30%。目前单碱基编辑器被广泛用于多种细胞系和模式生物中,包括人类遗传疾病的动物模型。
当前的CBE和ABE能够在各种序列背景下以及在包括哺乳动物在内的各种细胞类型和生物体中,在整个基因组的目标基因座上进行精准的定点突变,特定条件下还可以进行靶位点的随机突变。目前,单细胞编辑器还需要在编辑效率、特异性和体内递送能力上做进一步优化,并且开发活性可控的单碱基编辑器或使用外源性小分子可进一步提升其作为研究工具和潜在疗法的实用性。
As in CRISPR, the specific locus targeted with sgRNA must be followed by a PAM (protospacer adjacent motif) to allow for efficient Cas9 binding . In the structure of the Cas9 R-loop complex
, the eleven nucleotides furthest from the PAM on the nontarget strand are disordered, suggesting they are unencumbered and accessible for base editing.
Figure . A schematic showing R-loop formation by the base editors and the interaction between the cytidine deaminase enzyme and ssDNA.
Once the R-loop forms, the cytidine deaminase enzyme directly binds the target nucleotide (C) and chemically converts it to U. The resulting U:G mismatch is then processed by cellular DNA replication or repair, and resolves into a T:A base pair. The overall DNA transformation is therefore C:G to T:A.
Figure . Depiction of the initial, intermediate, and final DNA sequences involved in base editing, as well as a schematic of the chemical reaction being catalyzed by the deaminase enzyme.
单碱基编辑系统使用的注意事项
目前单碱基编辑器的多样性增加了其成功应用的可能性,这也让合理选择适合的单碱基编辑器更具有挑战性。选择单碱基编辑器时需要考虑,PAM的可用性,核苷酸序列信息,编辑窗口,产物纯度和DNA特异性等。