基于CRISPR/Cas系统的DNA碱基编辑研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-0311

摘要/Abstract

摘要：

基于CRISPR/Cas的基因编辑是近年发展起来的一项变革性生物技术。其过程包括在目标DNA位点引入双链断裂（double strand break,DSB）以及其后续的细胞修复。细胞修复DSB主要有两种方式：非同源末端连接（non-homologous end joining,NHEJ）以及同源重组介导的修复（homology-directed repair,HDR）。前者是大多数细胞修复DSB的主要方式,其特点在于修复简单、效率高但极易出错,往往会引发难以预测的核苷酸插入或删除。点突变是自然界中最常见的遗传突变类型,引起了超过半数的人类遗传疾病以及许多重要农艺性状变异。碱基编辑能够实现单个碱基的替换,既不需要引入DSB,又无需修复模板参与,具有高效、编辑结果可控等优点,在基因治疗、作物育种及生物技术研究等方面具有重大的应用潜能。自首个碱基编辑工具开发以来,碱基编辑相关技术得到快速发展及广泛应用。本文综述了目前DNA碱基编辑研究进展,重点阐述了碱基编辑器及其在编辑效率、精度以及特异性提高和编辑范围扩展等方面的最新进展以及仍存在的瓶颈,并展望其研究和应用前景。

关键词: 碱基编辑, 脱氨酶, 基因编辑, CRISPR, CRISPR/Cas

Abstract:

CRISPR/Cas-based genome editing has revolutionized biological technology,which is achieved by introducing DSB（double strand break）in target DNA sites followed by cellular DNA repair via non-homologous end joining（NHEJ）or homology-directed repair（HDR）. NHEJ is the main repair pathway in most of cells,it characterized as repair is simple,efficiency is high and it is extremely easy to have errors,and thus presents randomly and unpredictable insertions and deletions（indels）. Point mutations represent the most prevalent form of genetic mutations,which determine the many important agronomic traits in crop plants and cause most of known human genetic diseases. Base editing enables targeted single base substitutions of high efficiency and predicted editing outcomes with neither inducing DSBs in the genomes nor requiring donor templates. Based on its great applicable potentials in the gene therapy,crop breeding as well as basic research,base editing rapidly gains much attentions with numerous applications in agricultural as well as biological fields and considerable progresses having been made to broaden its capabilities. In this review,we summarize the current advances in DNA base editing,focus on improvements in editing efficiency,precision,specificity as well as editing scope,and discuss current limitations and future directions.

Key words: base editing, deaminase, gene editing, CRISPR, CRISPR/Cas

赖昕彤, 王柯岚, 由雨欣, 谭俊杰. 基于CRISPR/Cas系统的DNA碱基编辑研究进展[J]. 生物技术通报, 2022, 38(6): 1-12.

LAI Xin-tong, WANG Ke-lan, YOU Yu-xin, TAN Jun-jie. Recent Advances in CRISPR/Cas-based DNA Base Editing[J]. Biotechnology Bulletin, 2022, 38(6): 1-12.

图/表 3

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碱基编辑器名称 Base editor	结构组成 Architecture	编辑窗口 Editing window	序列偏好 Sequence preference	参考文献 Reference
BE3	rAPOBEC1-XTEN-nCas9-UGI	C₄- C₈	TC	^[10]
AID-BE3	hAID-XTEN-nCas9-UGI	C₃- C₈	None	^[23]
Target-AID	nCas9-linker-PmCDA1-UGI	C₂ - C₅	None	^[33-34]
CDA1-BE3	PmCDA1-XTEN-nCas9-UGI	C₁ - C₇	None	^[34]
CDA1Δ-BE3	PmCDA1Δ-nCas9-UGI	C₃ - C₄	None	^[34]
A3A-BE3	hA3A-XTEN-nCas9-UGI	C₄- C₈	None	^[38⇓-40]
eA3A-BE3	hA3A（N57G）-XTEN-nCas9-UGI	C₄- C₈	TC	^[38]
A3A∆-BE3	hA3AΔ-nCas9-UGI	C₅- C₆	None	^[35]
YE1-BE3	rAPOBEC1（W90Y/R126E）-XTEN-nCas9-UGI	C₅- C₇	TC	^[46]
YE2-BE3	rAPOBEC1（W90Y/R132E）-XTEN-nCas9-UGI	C₅- C₆	TC	^[46]
YEE-BE3	rAPOBEC1（W90Y/R126E/R132E）-XTEN-nCas9-UGI	C₅- C₆	TC	^[46]
SECURE-BE3	rAPOBEC1（R33A or R33A/K34A）-XTEN-nCas9-UGI	C₅- C₇	TC	^[80]
SaBE3	rAPOBEC1-XTEN-Sa nCas9-UGI	C₃- C₁₂	TC	^[46]
dCpf1-BE	rAPOBEC1-XTEN-dCpf1-UGI	C₈- C₁₃	TC	^[54]
BE-PLUS	GCN4（10×）-nCas9 scFv-rAPOBEC1-UGI	C₄- C₁₄	TC	^[48]
TAM	dCas9-linker-hAID（P182X）	C₄- C₈	None	^[53]
CRISPR-X	dCas9/MS2-linker-hAIDΔ	C_-50- C₅₀	None	^[36]
A3G-BE3	hA3G-XTEN-nCas9-UGI	C₄- C₈	CC	^[23]
eA3G-BE	hA3G-CTD-XTEN-nCas9-2*UGI	C₄- C₈	CC	^[41-42]
ABE7.10	TadA-linker-evoTadA-linker-nCas9	A₄- A₇	None	^[11]
ABE7.10^F148A	TadA^F148A-evoTadA^F148A-linker-nCas9	A₅	None	^[47]
dCasMINI-ABE	TadA-linker-evoTadA-linker- dCasMINI	A₃- A₄	None	^[55]
CP-ABEs	TadA -linker-evoTadA -linker-CP-nCas9s	A₄- A₁₂	None	^[57]

碱基编辑器名称 Base editor	结构组成 Architecture	编辑窗口 Editing window	序列偏好 Sequence preference	参考文献 Reference
BE3	rAPOBEC1-XTEN-nCas9-UGI	C₄- C₈	TC	^[10]
AID-BE3	hAID-XTEN-nCas9-UGI	C₃- C₈	None	^[23]
Target-AID	nCas9-linker-PmCDA1-UGI	C₂ - C₅	None	^[33-34]
CDA1-BE3	PmCDA1-XTEN-nCas9-UGI	C₁ - C₇	None	^[34]
CDA1Δ-BE3	PmCDA1Δ-nCas9-UGI	C₃ - C₄	None	^[34]
A3A-BE3	hA3A-XTEN-nCas9-UGI	C₄- C₈	None	^[38⇓-40]
eA3A-BE3	hA3A（N57G）-XTEN-nCas9-UGI	C₄- C₈	TC	^[38]
A3A∆-BE3	hA3AΔ-nCas9-UGI	C₅- C₆	None	^[35]
YE1-BE3	rAPOBEC1（W90Y/R126E）-XTEN-nCas9-UGI	C₅- C₇	TC	^[46]
YE2-BE3	rAPOBEC1（W90Y/R132E）-XTEN-nCas9-UGI	C₅- C₆	TC	^[46]
YEE-BE3	rAPOBEC1（W90Y/R126E/R132E）-XTEN-nCas9-UGI	C₅- C₆	TC	^[46]
SECURE-BE3	rAPOBEC1（R33A or R33A/K34A）-XTEN-nCas9-UGI	C₅- C₇	TC	^[80]
SaBE3	rAPOBEC1-XTEN-Sa nCas9-UGI	C₃- C₁₂	TC	^[46]
dCpf1-BE	rAPOBEC1-XTEN-dCpf1-UGI	C₈- C₁₃	TC	^[54]
BE-PLUS	GCN4（10×）-nCas9 scFv-rAPOBEC1-UGI	C₄- C₁₄	TC	^[48]
TAM	dCas9-linker-hAID（P182X）	C₄- C₈	None	^[53]
CRISPR-X	dCas9/MS2-linker-hAIDΔ	C_-50- C₅₀	None	^[36]
A3G-BE3	hA3G-XTEN-nCas9-UGI	C₄- C₈	CC	^[23]
eA3G-BE	hA3G-CTD-XTEN-nCas9-2*UGI	C₄- C₈	CC	^[41-42]
ABE7.10	TadA-linker-evoTadA-linker-nCas9	A₄- A₇	None	^[11]
ABE7.10^F148A	TadA^F148A-evoTadA^F148A-linker-nCas9	A₅	None	^[47]
dCasMINI-ABE	TadA-linker-evoTadA-linker- dCasMINI	A₃- A₄	None	^[55]
CP-ABEs	TadA -linker-evoTadA -linker-CP-nCas9s	A₄- A₁₂	None	^[57]