Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (1): 67-85.DOI: 10.12211/2096-8280.2022-047
• Invited Review • Previous Articles Next Articles
Xiaolong TENG, Shuobo SHI
Received:
2022-09-01
Revised:
2022-10-28
Online:
2023-03-07
Published:
2023-02-28
Contact:
Shuobo SHI
滕小龙, 史硕博
通讯作者:
史硕博
作者简介:
基金资助:
CLC Number:
Xiaolong TENG, Shuobo SHI. Optimization and development of CRISPR/Cas9 systems for genome editing[J]. Synthetic Biology Journal, 2023, 4(1): 67-85.
滕小龙, 史硕博. CRISPR/Cas9系统在基因组编辑中的优化与发展[J]. 合成生物学, 2023, 4(1): 67-85.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2022-047
Fig. 1 Gene editing mechanism of CRISPR/Cas9 systems(NHEJ-mediated fragment deletion/insertion, HDR-mediated gene editing and MMEJ-mediated gene editing are shown diagrammatically from left and middle to right)
变体及直系同源 | 改造位点或方式 | PAM | 特性 | 参考文献 |
---|---|---|---|---|
SpCas9 | 原生酿脓链球菌Cas9 | NGG | 1368个氨基酸 | [ |
dCas9 | D10A,H840A | NGG | 核酸酶活性失活 | [ |
nCas9(D10A) | D10A | NGG | 切口酶,非靶向链切割活性失活 | [ |
nCas9(H840A) | H840A | NGG | 切口酶,靶向链切割活性失活 | [ |
SpCas9NG | R1335V,L1111R,D1135V,G1218R,E1219F,A1322R,T1337R | NG | PAM改变;通过Cas9理性设计获得 | [ |
VRERSpCas9 | D1135A,G1218R,R1335E,T1337R | NGCG | PAM改变;通过细菌选择性定向进化获得 | [ |
VQRSpCas9 | D1135V,R1335Q,T1337R | NGAN,NGNG | PAM改变;通过细菌选择性定向进化获得 | [ |
EQRSpCas9 | D1335E,R1335Q,T1337R | NGAG | PAM改变;通过细菌选择性定向进化获得 | [ |
xCas9 | A262T,R324L,S409I,E480K,E543D,M694I,E1219V | NG,GAA,GAT | PAM改变;通过噬菌体辅助持续进化获得 | [ |
SpG | D1135L,S1136W,G1218K,E1219Q,R1335Q,T1337R | NGN | PAM改变;通过SpCas9理性设计获得 | [ |
SpRY | SpG,A61R,L1111R,A1322R,N1317R,R1333P | NRN,NYN | PAM改变;通过对SpG进一步理性设计获得 | [ |
evoCas9 | M495V, Y515N, K526E, R661Q | NGG | 保真性提高,编辑效率接近天然SpCas9 | [ |
fCas9 | SpdCas9与FokI融合 | NGG | 保真性提高;通过将FokI核酸酶与dCas9融合获得 | [ |
StCas9 | 原生嗜热链球菌Cas9 | NNAGAAW | 1121个氨基酸 | [ |
NmCas9 | 原生脑膜炎奈瑟菌Cas9 | NNNNGATT | 1082个氨基酸 | [ |
SaCas9 | 原生金黄色葡萄球菌Cas9 | NNGRRT | 1053个氨基酸;基因组编辑效率与SpCas9相当 | [ |
CjCas9 | 原生曲状杆菌Cas9 | NNNVRYM | 984个氨基酸 | [ |
Table 1 Major Cas9 variants and orthologues as well as their features
变体及直系同源 | 改造位点或方式 | PAM | 特性 | 参考文献 |
---|---|---|---|---|
SpCas9 | 原生酿脓链球菌Cas9 | NGG | 1368个氨基酸 | [ |
dCas9 | D10A,H840A | NGG | 核酸酶活性失活 | [ |
nCas9(D10A) | D10A | NGG | 切口酶,非靶向链切割活性失活 | [ |
nCas9(H840A) | H840A | NGG | 切口酶,靶向链切割活性失活 | [ |
SpCas9NG | R1335V,L1111R,D1135V,G1218R,E1219F,A1322R,T1337R | NG | PAM改变;通过Cas9理性设计获得 | [ |
VRERSpCas9 | D1135A,G1218R,R1335E,T1337R | NGCG | PAM改变;通过细菌选择性定向进化获得 | [ |
VQRSpCas9 | D1135V,R1335Q,T1337R | NGAN,NGNG | PAM改变;通过细菌选择性定向进化获得 | [ |
EQRSpCas9 | D1335E,R1335Q,T1337R | NGAG | PAM改变;通过细菌选择性定向进化获得 | [ |
xCas9 | A262T,R324L,S409I,E480K,E543D,M694I,E1219V | NG,GAA,GAT | PAM改变;通过噬菌体辅助持续进化获得 | [ |
SpG | D1135L,S1136W,G1218K,E1219Q,R1335Q,T1337R | NGN | PAM改变;通过SpCas9理性设计获得 | [ |
SpRY | SpG,A61R,L1111R,A1322R,N1317R,R1333P | NRN,NYN | PAM改变;通过对SpG进一步理性设计获得 | [ |
evoCas9 | M495V, Y515N, K526E, R661Q | NGG | 保真性提高,编辑效率接近天然SpCas9 | [ |
fCas9 | SpdCas9与FokI融合 | NGG | 保真性提高;通过将FokI核酸酶与dCas9融合获得 | [ |
StCas9 | 原生嗜热链球菌Cas9 | NNAGAAW | 1121个氨基酸 | [ |
NmCas9 | 原生脑膜炎奈瑟菌Cas9 | NNNNGATT | 1082个氨基酸 | [ |
SaCas9 | 原生金黄色葡萄球菌Cas9 | NNGRRT | 1053个氨基酸;基因组编辑效率与SpCas9相当 | [ |
CjCas9 | 原生曲状杆菌Cas9 | NNNVRYM | 984个氨基酸 | [ |
功能 | 效应蛋白或gRNA工程 | 系统 | 特征 | 参考文献 |
---|---|---|---|---|
表达调控 | 无 | dCas9 | 原核生物基因抑制表达,真核生物基因抑制效率低 | [ |
KRAB,MXI1,TUP1抑制结构域 | dCas9 | 可在真核生物中抑制基因表达 | [ | |
KRAB-MeCP1融合抑制结构域 | dCas9 | 在哺乳动物细胞中高效抑制目的基因表达 | [ | |
p65,VP64激活结构域 | dCas9 | 激活基因表达,效率较低 | [ | |
VP64-p65-Rta(VPR)融合激活结构域 | dCas9 | 与VP64相比提高了22~320倍;多重基因激活表达;刺激iPSCs神经元分化 | [ | |
gRNA工程;gRNA上携带携带两个MS2发夹二级结构 | dCas9 | 每个发夹结构招募一个MS2-p65-HSF1融合激活蛋白;与VP64相比,使10个靶基因上调2倍以上 | [ | |
SPY系统(包括SpyTag和SpyCatcher)和Med2激活结构域 | dCas9 | 招募多拷贝SpyCatcher和Med2融合蛋白使酿酒酵母目的基因表达提高34.9倍 | [ | |
SunTag系统(包括GCN4肽和抗GCN4抗体的单链可变片段scFv)和MIG1抑制结构域 | dCpf1 | 招募多拷贝ScFv与MIG1融合蛋白使酿酒酵母目的基因受到95%的抑制 | [ | |
表观基因组编辑 | KRAB-DNA甲基化酶催化结构域融合蛋白 | dCas9 | 融合抑制因子和甲基化酶;造成目的基因78%的稳定沉默 | [ |
两侧分别融合KRAB和Dnmt3A-Dnmt3L融合DNA甲基化酶结构域 | dCas9 | 在干细胞分裂分化为神经细胞后仍维持DNA甲基化和基因抑制 | [ | |
TET1甲基胞嘧啶双加氧酶1催化结构域 | dCas9 | dCas9- TET1靶向至BRCA1基因启动子后成功地上调其表达水平 | [ | |
人类组蛋白乙酰基转移酶p300催化核心 | dCas9 | 催化组蛋白H3 第27位赖氨酸残基的乙酰基化;基因表达上调 | [ | |
单碱基编辑 | 胞嘧啶脱氨酶,腺嘌呤脱氨酶 | nCas9 | 在不造成DNA双链断裂的条件下使目的碱基产生C:G与T:A或A:T与G:C的碱基对替换 | [ |
CBE-UGI融合蛋白 | nCas9 | UGI能够防止胞嘧啶C脱氨后的尿嘧啶U被切除,从而显著提高了CBEs的编辑效率 | [ | |
单链DNA结合结构域ssDBD融合到胞嘧啶脱氨酶与nCas9之间 | nCas9 | 通过延长ssDNA的暴露时间极大地提高CBEs的编辑效率;使CBEs的编辑窗口范围得到了提高 | [ | |
组合nCas9、胞嘧啶脱氨酶和尿嘧啶-DNA糖基化酶 | nCas9 | 首次实现大肠杆菌中C到A 87.2%和哺乳动物细胞C到G 5.3%至53.0%的编辑效率 | [ | |
先导编辑 | 通过gRNA工程改造的pegRNA;与逆转录酶融合 | nCas9 | 改变pegRNA可实现精确的碱基替换、基因敲除和插入 | [ |
通过gRNA工程改造的pegRNA;与逆转录酶和Rad51融合 | nCas9 | 融合单链DNA结合蛋白Rad51使PE2的编辑效率最高提升2.6倍 | [ |
Table 2 Functional optimization through fusion expression of effector proteins with CRISPR/Cas9 system
功能 | 效应蛋白或gRNA工程 | 系统 | 特征 | 参考文献 |
---|---|---|---|---|
表达调控 | 无 | dCas9 | 原核生物基因抑制表达,真核生物基因抑制效率低 | [ |
KRAB,MXI1,TUP1抑制结构域 | dCas9 | 可在真核生物中抑制基因表达 | [ | |
KRAB-MeCP1融合抑制结构域 | dCas9 | 在哺乳动物细胞中高效抑制目的基因表达 | [ | |
p65,VP64激活结构域 | dCas9 | 激活基因表达,效率较低 | [ | |
VP64-p65-Rta(VPR)融合激活结构域 | dCas9 | 与VP64相比提高了22~320倍;多重基因激活表达;刺激iPSCs神经元分化 | [ | |
gRNA工程;gRNA上携带携带两个MS2发夹二级结构 | dCas9 | 每个发夹结构招募一个MS2-p65-HSF1融合激活蛋白;与VP64相比,使10个靶基因上调2倍以上 | [ | |
SPY系统(包括SpyTag和SpyCatcher)和Med2激活结构域 | dCas9 | 招募多拷贝SpyCatcher和Med2融合蛋白使酿酒酵母目的基因表达提高34.9倍 | [ | |
SunTag系统(包括GCN4肽和抗GCN4抗体的单链可变片段scFv)和MIG1抑制结构域 | dCpf1 | 招募多拷贝ScFv与MIG1融合蛋白使酿酒酵母目的基因受到95%的抑制 | [ | |
表观基因组编辑 | KRAB-DNA甲基化酶催化结构域融合蛋白 | dCas9 | 融合抑制因子和甲基化酶;造成目的基因78%的稳定沉默 | [ |
两侧分别融合KRAB和Dnmt3A-Dnmt3L融合DNA甲基化酶结构域 | dCas9 | 在干细胞分裂分化为神经细胞后仍维持DNA甲基化和基因抑制 | [ | |
TET1甲基胞嘧啶双加氧酶1催化结构域 | dCas9 | dCas9- TET1靶向至BRCA1基因启动子后成功地上调其表达水平 | [ | |
人类组蛋白乙酰基转移酶p300催化核心 | dCas9 | 催化组蛋白H3 第27位赖氨酸残基的乙酰基化;基因表达上调 | [ | |
单碱基编辑 | 胞嘧啶脱氨酶,腺嘌呤脱氨酶 | nCas9 | 在不造成DNA双链断裂的条件下使目的碱基产生C:G与T:A或A:T与G:C的碱基对替换 | [ |
CBE-UGI融合蛋白 | nCas9 | UGI能够防止胞嘧啶C脱氨后的尿嘧啶U被切除,从而显著提高了CBEs的编辑效率 | [ | |
单链DNA结合结构域ssDBD融合到胞嘧啶脱氨酶与nCas9之间 | nCas9 | 通过延长ssDNA的暴露时间极大地提高CBEs的编辑效率;使CBEs的编辑窗口范围得到了提高 | [ | |
组合nCas9、胞嘧啶脱氨酶和尿嘧啶-DNA糖基化酶 | nCas9 | 首次实现大肠杆菌中C到A 87.2%和哺乳动物细胞C到G 5.3%至53.0%的编辑效率 | [ | |
先导编辑 | 通过gRNA工程改造的pegRNA;与逆转录酶融合 | nCas9 | 改变pegRNA可实现精确的碱基替换、基因敲除和插入 | [ |
通过gRNA工程改造的pegRNA;与逆转录酶和Rad51融合 | nCas9 | 融合单链DNA结合蛋白Rad51使PE2的编辑效率最高提升2.6倍 | [ |
宿主细胞 | 多重CRISPR策略 | 靶向目标数量 | 应用/概念验证 | 参考文献 |
---|---|---|---|---|
哺乳动物细胞 | 2个间隔区通过天然CRISPR序列自我加工 | 2个靶标(1个基因) | 对1个靶基因敲除,效率为1.6% | [ |
酿酒酵母 | Casy4核酸酶加工 | 3个基因 | 同时对3个不同启动子进行激活,使得3个报告基因荧光强度提升了2倍 | [ |
酿酒酵母 | 基于Csy4核酸酶加工的多gRNA快速组装 | 12个靶标(3个基因) | 通过12个gRNA靶向使得3个报告基因荧光强度分别被抑制了92%、81%和95% | [ |
酿酒酵母 | 基于tRNA转录本切割机制的多gRNA表达系统 | 8个基因 | 通过两轮8个基因的敲除获得了30倍的游离脂肪酸产量 | [ |
酿酒酵母 | 多个gRNA表达盒通过pol Ⅲ启动子启动 | 3个基因 | 将适配子与gRNA融合进行多基因调控,获得了不同的紫罗兰素生物合成产物 | [ |
酿酒酵母 | 基于Csy4核酸酶加工的多gRNA快速组装策略及多Cas9正交组合调控 | 3个基因 | 通过组合调控使两个报告基因表达分别被抑制和激活5倍,同时以95%的效率敲除第三个基因 | [ |
酿酒酵母 | 通过质粒表达多个gRNA表达盒以及截短gRNA用于调控 | 3个基因 | 通过对三个基因分别进行编辑、激活和抑制获得了α-檀香烯2.66倍的产量 | [ |
马克思克鲁维酵母(Kluyveromyces marxianus) | 基于tRNA转录本切割机制的多gRNA表达系统 | 6个靶标(4个基因) | 通过对4个基因进行调控,使乙酸乙酯的产量提升了3.8倍 | [ |
大肠杆菌 | 多个gRNA表达盒通过质粒表达 | 3个基因 | 通过对3个基因进行组合调控得到了2.3倍苹果酸产量菌株 | [ |
大肠杆菌 | 多个gRNA表达盒通过质粒表达 | 3个基因 | 对苹果酸合成途径基因进行组合调控后获得了2.3倍苹果酸产量菌株 | [ |
枯草芽孢杆菌(Bacillus subtilis) | 多个gRNA表达盒整合至基因组 | 3个基因 | 通过多基因动态组合调控提高了N-乙酰葡糖胺产量 | [ |
天蓝色链霉菌(Streptomyces coelicolor) | 多个gRNA表达盒与dCas9在质粒上表达 | 4个基因 | 通过对4个靶基因同时进行抑制,使其mRNA表达量降为对照的2%~32% | [ |
Table 3 Research on the applications of the multiplexed CRISPR/Cas9 system
宿主细胞 | 多重CRISPR策略 | 靶向目标数量 | 应用/概念验证 | 参考文献 |
---|---|---|---|---|
哺乳动物细胞 | 2个间隔区通过天然CRISPR序列自我加工 | 2个靶标(1个基因) | 对1个靶基因敲除,效率为1.6% | [ |
酿酒酵母 | Casy4核酸酶加工 | 3个基因 | 同时对3个不同启动子进行激活,使得3个报告基因荧光强度提升了2倍 | [ |
酿酒酵母 | 基于Csy4核酸酶加工的多gRNA快速组装 | 12个靶标(3个基因) | 通过12个gRNA靶向使得3个报告基因荧光强度分别被抑制了92%、81%和95% | [ |
酿酒酵母 | 基于tRNA转录本切割机制的多gRNA表达系统 | 8个基因 | 通过两轮8个基因的敲除获得了30倍的游离脂肪酸产量 | [ |
酿酒酵母 | 多个gRNA表达盒通过pol Ⅲ启动子启动 | 3个基因 | 将适配子与gRNA融合进行多基因调控,获得了不同的紫罗兰素生物合成产物 | [ |
酿酒酵母 | 基于Csy4核酸酶加工的多gRNA快速组装策略及多Cas9正交组合调控 | 3个基因 | 通过组合调控使两个报告基因表达分别被抑制和激活5倍,同时以95%的效率敲除第三个基因 | [ |
酿酒酵母 | 通过质粒表达多个gRNA表达盒以及截短gRNA用于调控 | 3个基因 | 通过对三个基因分别进行编辑、激活和抑制获得了α-檀香烯2.66倍的产量 | [ |
马克思克鲁维酵母(Kluyveromyces marxianus) | 基于tRNA转录本切割机制的多gRNA表达系统 | 6个靶标(4个基因) | 通过对4个基因进行调控,使乙酸乙酯的产量提升了3.8倍 | [ |
大肠杆菌 | 多个gRNA表达盒通过质粒表达 | 3个基因 | 通过对3个基因进行组合调控得到了2.3倍苹果酸产量菌株 | [ |
大肠杆菌 | 多个gRNA表达盒通过质粒表达 | 3个基因 | 对苹果酸合成途径基因进行组合调控后获得了2.3倍苹果酸产量菌株 | [ |
枯草芽孢杆菌(Bacillus subtilis) | 多个gRNA表达盒整合至基因组 | 3个基因 | 通过多基因动态组合调控提高了N-乙酰葡糖胺产量 | [ |
天蓝色链霉菌(Streptomyces coelicolor) | 多个gRNA表达盒与dCas9在质粒上表达 | 4个基因 | 通过对4个靶基因同时进行抑制,使其mRNA表达量降为对照的2%~32% | [ |
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