合成生物学 ›› 2023, Vol. 4 ›› Issue (1): 86-101.DOI: 10.12211/2096-8280.2022-040
梁丽亚, 刘嵘明
收稿日期:
2022-07-18
修回日期:
2022-09-28
出版日期:
2023-02-28
发布日期:
2023-03-07
通讯作者:
刘嵘明
作者简介:
基金资助:
Liya LIANG, Rongming LIU
Received:
2022-07-18
Revised:
2022-09-28
Online:
2023-02-28
Published:
2023-03-07
Contact:
Rongming LIU
摘要:
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein是细菌或古细菌特有的一种获得性免疫系统,自从研究人员将其改造为基因编辑工具之后,CRISPR/Cas系统高效的基因编辑能力对生命科学、生物工程、生物医药、食品及农业科学等多个领域引发了革命性的影响。然而,研究者们发现CRISPR/Cas系统存在脱靶效应、PAM位点识别范围有限等问题,限制了CRISPR/Cas的应用。为了解决这些问题,针对Cas蛋白进行工程化改造已经成为了优化及开发CRISPR/Cas系统的重要策略。本文以Ⅱ类CRISPR/Cas系统中靶向DNA的CRISPR/Cas9以及CRISPR/Cas12a为例,聚焦近年来针对Cas9和Cas12a蛋白的优化改造方法及相关进展进行系统阐述总结,包括Cas蛋白的工程化改造提升基因编辑特异性及改变PAM识别能力,以CRISPR/Cas系统作为基因定位工具开发新功能,结合外源蛋白调控CRISPR/Cas系统功能。这些工作开发了系列高特异性、高精度的CRISPR/Cas系统,极大地扩展了CRISPR/Cas系统的功能及适用范围,为CRISPR/Cas系统的多领域应用做出了重要贡献。
中图分类号:
梁丽亚, 刘嵘明. 靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造[J]. 合成生物学, 2023, 4(1): 86-101.
Liya LIANG, Rongming LIU. Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems[J]. Synthetic Biology Journal, 2023, 4(1): 86-101.
图1 Streptococcus pyogenes Cas9与crRNA和靶DNA复合物的晶体结构 (PDB: 4OO8) 和Acidaminococcus sp. Cas12a与crRNA和靶DNA复合物的晶体结构 (PDB: 5B43)[26]
Fig. 1 Crystal structures of the complexes of Streptococcus pyogenes Cas9 (PDB: 4OO8) and Acidaminococcus sp. Cas12a (PDB: 5B43)with guide RNA and target DNA [26]
名称 | 突变 | 改造的结构域 | PAM | 精度最高 提升倍数 | 参考文献 |
---|---|---|---|---|---|
SpCas9-HF1 | N497A/R661A/Q695A/Q926A | REC3, RuvC3 | NGG | 19 | [ |
eSpCas9(1.1) | K848A/K1003A/R1060A | HNH, RuvC3 (nt-groove) | NGG | 19 | [ |
HypaCas9 | N692A/M694A/Q695A/H698A | REC3 | NGG | 19 | [ |
SuperFi-Cas9 | Y1010D/Y1013D/Y1016D/V1018D/R1019D/Q1027D/K1031D | RuvC | NGG | 4.06② | [ |
hscCas9-v1.2 | N14A/R765A/R447A/S845D | REC1, RuvC, HNH | NGG | 1.67 | [ |
SaCas9-HF | R245A/N413A/N419A/R654A | REC, RuvC3 | NNGRRT | 2 | [ |
enAsCas12a-HF1 | N282A① | REC1 | TTYN, VTTV, TRTV | 1.89 | [ |
表1 基于蛋白结构的理性或半理性设计提高Cas蛋白特异性
Table 1 Engineering Cas proteins for improved on-target specificity using rational/semi-rational design based on protein crystal structures
名称 | 突变 | 改造的结构域 | PAM | 精度最高 提升倍数 | 参考文献 |
---|---|---|---|---|---|
SpCas9-HF1 | N497A/R661A/Q695A/Q926A | REC3, RuvC3 | NGG | 19 | [ |
eSpCas9(1.1) | K848A/K1003A/R1060A | HNH, RuvC3 (nt-groove) | NGG | 19 | [ |
HypaCas9 | N692A/M694A/Q695A/H698A | REC3 | NGG | 19 | [ |
SuperFi-Cas9 | Y1010D/Y1013D/Y1016D/V1018D/R1019D/Q1027D/K1031D | RuvC | NGG | 4.06② | [ |
hscCas9-v1.2 | N14A/R765A/R447A/S845D | REC1, RuvC, HNH | NGG | 1.67 | [ |
SaCas9-HF | R245A/N413A/N419A/R654A | REC, RuvC3 | NNGRRT | 2 | [ |
enAsCas12a-HF1 | N282A① | REC1 | TTYN, VTTV, TRTV | 1.89 | [ |
名称 | 突变 | 改造的结构域 | PAM | 精度最高 提升倍数 | 参考文献 |
---|---|---|---|---|---|
evoCas9 | M495V/Y515N/K526E/H698Q | REC3 | NGG | 9 | [ |
HiFi Cas9 | R691A | REC3 | NGG | 2.46 | [ |
xCas9-3.7 | A262T/R324L/S409I/E480K/E543D/M694I/E1219V① | REC1, REC2, REC3, PI | NG, NNG, GAA, GAT and CAA | 0.29 | [ |
Sniper-Cas9 | F539S/M763I/K890N | REC3, RuvC2, HNH | NGG | 8.47 | [ |
Opti-SpCas9 | R661A/K1003H | REC3, RuvC3 | NGG | 5 | [ |
AsCas12a-K949A | K949A | BH | TTTV | 0.23 | [ |
M44 | TX_Cas12a的REC1结构域替换 | REC1 | TTTN | 24 | [ |
表2 基于模式菌株的高效筛选方法提高Cas蛋白特异性
Table 2 Engineering Cas protein for improved on-target specificity based on efficient selection in model strains
名称 | 突变 | 改造的结构域 | PAM | 精度最高 提升倍数 | 参考文献 |
---|---|---|---|---|---|
evoCas9 | M495V/Y515N/K526E/H698Q | REC3 | NGG | 9 | [ |
HiFi Cas9 | R691A | REC3 | NGG | 2.46 | [ |
xCas9-3.7 | A262T/R324L/S409I/E480K/E543D/M694I/E1219V① | REC1, REC2, REC3, PI | NG, NNG, GAA, GAT and CAA | 0.29 | [ |
Sniper-Cas9 | F539S/M763I/K890N | REC3, RuvC2, HNH | NGG | 8.47 | [ |
Opti-SpCas9 | R661A/K1003H | REC3, RuvC3 | NGG | 5 | [ |
AsCas12a-K949A | K949A | BH | TTTV | 0.23 | [ |
M44 | TX_Cas12a的REC1结构域替换 | REC1 | TTTN | 24 | [ |
名称 | 突变 | 改造的结构域 | PAM范围变化 | 参考文献 |
---|---|---|---|---|
SpCas9① | NGG | [ | ||
xCas9-3.7 | A262T/R324L/S409I/E480K/E543D/M694I/E1219V | REC1, REC2, REC3, PI | NG, NNG, GAA, GAT and CAA | [ |
SaCas9① | NNGRRT② | [ | ||
SaCas9 KKH | E782K/N968K/R1015H | PI | NNNRRT | [ |
v42-wt | SaCas9 上PI结构域的13个保守氨基酸序列被替换 | PI | NNVRRN② | [ |
AsCas12a/LbCas12a① | TTTV | [ | ||
AsCas12a | S542R/K607R | WED, PI | TYCV② | [ |
AsCas12a | S542R/K548V/N552R | WED, PI | TATV | [ |
LbCas12a | G532R/K595R | WED, PI | TYCV | [ |
LbCas12a | G532R/K538V/Y542R | WED, PI | TATV | [ |
表3 工程化改造Cas蛋白改变PAM位点的碱基组成
Table 3 Engineering Cas proteins to change the nucleotide sequences of PAM
名称 | 突变 | 改造的结构域 | PAM范围变化 | 参考文献 |
---|---|---|---|---|
SpCas9① | NGG | [ | ||
xCas9-3.7 | A262T/R324L/S409I/E480K/E543D/M694I/E1219V | REC1, REC2, REC3, PI | NG, NNG, GAA, GAT and CAA | [ |
SaCas9① | NNGRRT② | [ | ||
SaCas9 KKH | E782K/N968K/R1015H | PI | NNNRRT | [ |
v42-wt | SaCas9 上PI结构域的13个保守氨基酸序列被替换 | PI | NNVRRN② | [ |
AsCas12a/LbCas12a① | TTTV | [ | ||
AsCas12a | S542R/K607R | WED, PI | TYCV② | [ |
AsCas12a | S542R/K548V/N552R | WED, PI | TATV | [ |
LbCas12a | G532R/K595R | WED, PI | TYCV | [ |
LbCas12a | G532R/K538V/Y542R | WED, PI | TATV | [ |
名称 | 突变 | 改造的结构域 | PAM范围变化 | 参考文献 |
---|---|---|---|---|
SpCas9① | NGG | [ | ||
SpCas9 VQR | D1135V/R1335Q/T1337R | WED, PI | NGAN NGCG | [ |
SpCas9 EQR | D1135E/R1335Q/T1337R | WED, PI | NGAG | [ |
SpCas9 VRER | D1135V/G1218R/R1335E/T1337R | WED, PI | NGCG | [ |
Sp-St3Cas9 | SpCas9 与St3Cas9的PI结构域替换 | PI | NGGNG | [ |
SpCas9-NG | R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R | WED, PI | NG | [ |
SpG | D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R | WED, PI | NGN | [ |
SpRY | SpG(A61R/L1111R/A1322R/ /N1317R/ R1333P) | BH, WED, PI | NRN | [ |
FnCas9① | NGG | [ | ||
RHA FnCas9 | E1369R/E1449H/R1556A | WED, PI | YG② | [ |
表4 工程化改造Cas蛋白增加或减少PAM位点的碱基数量
Table 4 Engineering Cas proteins to increase or decrease the nucleotide number of PAM
名称 | 突变 | 改造的结构域 | PAM范围变化 | 参考文献 |
---|---|---|---|---|
SpCas9① | NGG | [ | ||
SpCas9 VQR | D1135V/R1335Q/T1337R | WED, PI | NGAN NGCG | [ |
SpCas9 EQR | D1135E/R1335Q/T1337R | WED, PI | NGAG | [ |
SpCas9 VRER | D1135V/G1218R/R1335E/T1337R | WED, PI | NGCG | [ |
Sp-St3Cas9 | SpCas9 与St3Cas9的PI结构域替换 | PI | NGGNG | [ |
SpCas9-NG | R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R | WED, PI | NG | [ |
SpG | D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R | WED, PI | NGN | [ |
SpRY | SpG(A61R/L1111R/A1322R/ /N1317R/ R1333P) | BH, WED, PI | NRN | [ |
FnCas9① | NGG | [ | ||
RHA FnCas9 | E1369R/E1449H/R1556A | WED, PI | YG② | [ |
名称 | CRISPR/Cas | Base editor | PE |
---|---|---|---|
Class/Type | Type Ⅱ | Type Ⅱ | Type Ⅱ |
靶向序列长度/bp | 19~23 | 15~20 | 8~15 |
DNA编辑方式 | 有DSB的HDR | 无DSB的碱基替换 | 无DSB的HDR |
On-target | 高 | 较高 | 低 |
Off-target | 较低 | 低 | 低 |
多位点编辑 | 可达9位点[ | 可达31位点[ | 可达3位点[ |
应用范围 | 人体、动物、植物、微生物 | 人体、动物、植物、微生物 | 人体、动物、植物、微生物 |
表5 CRISPR/Cas系统与Base editor、PE的比较
Table 5 Comparison among CRISPR/Cas, base editor, and PE
名称 | CRISPR/Cas | Base editor | PE |
---|---|---|---|
Class/Type | Type Ⅱ | Type Ⅱ | Type Ⅱ |
靶向序列长度/bp | 19~23 | 15~20 | 8~15 |
DNA编辑方式 | 有DSB的HDR | 无DSB的碱基替换 | 无DSB的HDR |
On-target | 高 | 较高 | 低 |
Off-target | 较低 | 低 | 低 |
多位点编辑 | 可达9位点[ | 可达31位点[ | 可达3位点[ |
应用范围 | 人体、动物、植物、微生物 | 人体、动物、植物、微生物 | 人体、动物、植物、微生物 |
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