CRISPR-Cas系统的小型化研究进展

doi:10.12211/2096-8280.2023-068

摘要/Abstract

摘要：

CRISPR-Cas基因编辑技术由于其简便性和高效性，已被广泛应用于生物学、医学、农学等领域的基础与应用研究。目前广泛使用的Cas核酸酶均具有较大的分子量（通常大于1000个氨基酸），而广泛应用于基因治疗中的腺相关病毒（AAV）载体的承载容量却十分有限，在容纳CRISPR核酸酶与gRNA的编码序列之余往往难以承载更多其它功能元件，如碱基编辑、转录调控、多基因编辑等相应元件，这严重限制了其在基因治疗等领域的应用。使用紧凑型Cas蛋白变体的CRISPR-Cas系统可能有助于用AAV产生和传递基因组编辑和调节工具到人类细胞。因此，小型化的CRISPR-Cas系统开发是解决这一技术难题的重要途径，本文主要概括了基于Cas9、Cas12和Cas13蛋白系统在小型化方面的研究进展，包括筛选新型Cas蛋白、缩减蛋白结构域以及引导RNA的改造等，旨在为开发微型精准基因编辑和调控工具提供新思路。目前小型化的CRISPR-Cas系统的局限性主要体现在蛋白分子量的大小和基因编辑的效率、特异性不可兼得上，在未来的研究中若能解决这一问题，获得更小化的结构域，我相信我们不仅能够优化该系统在体内的传递，更有望为临床带来高效率且低损害的治疗方法。

关键词: CRISPR-Cas系统, 小型化Cas蛋白, Cas蛋白工程化改造

Abstract:

The CRISPR-Cas gene editing technology has revolutionized the fields of biology, medicine, and agronomy due to its remarkable simplicity and efficiency. Laboratory-developed tools, such as the widely recognized CRISPR-Cas9, have played a pivotal role in addressing a multitude of genetic diseases. By harnessing the targeted nucleic acid capabilities of the CRISPR-Cas system, researchers have successfully integrated various functionalities into Cas proteins, including fluorescent markers, transcriptional regulatory proteins, and base editing components. This has unlocked new possibilities, including chromosome imaging, transcriptional regulation, and precise base editing. Currently, Cas nucleases with substantial molecular weights, often exceeding 1000 amino acids, are the norm. In contrast, adeno-associated virus (AAV) vectors, which are extensively utilized in gene therapy, have limited capacity to accommodate additional functional components beyond the coding sequences of CRISPR nucleases and guide RNAs (gRNAs). This limitation severely constrains their utility in gene therapy and other applications. As a result, a significant focus of research has been placed on the miniaturization of CRISPR tools, making them compact enough to align with current delivery methods. Compact Cas protein variants within CRISPR-Cas systems hold the potential to create and deliver genome editing and regulatory tools into human cells using AAV. Hence, the development of miniaturized CRISPR-Cas systems represents a crucial avenue to address this technical challenge. This paper provides a comprehensive review of research progress in miniaturizing key proteins within two classes of systems: Cas9 and Cas12, which target DNA, and Cas13, which targets RNA. This review encompasses the screening of novel Cas proteins, the reduction of protein structural domains, and the modification of guide RNAs, all with the intention of presenting innovative ideas for the further advancement of compact, precise gene editing, and regulatory tools. The miniaturization of CRISPR-Cas systems is a critical step toward unlocking their full potential in various fields, including biomedicine, agriculture, and basic research. As researchers continue to explore and refine these compact gene editing and regulatory tools, we can expect significant advancements in our ability to manipulate and understand genetic information. This ongoing progress promises to have a profound impact on the future of science and technology. At present, the limitations of the miniaturized CRISPR-Cas system are mainly reflected in the size of protein molecular weight and the efficiency and specificity of gene editing. If we can solve this problem and obtain a smaller structure in future research, I believe that we can not only optimize the transmission of the system in the body, but also hope to bring high-efficiency and low-damage treatment methods to the clinic.

Key words: CRISPR-Cas systems, miniaturized Cas proteins, Cas protein engineering modification

中图分类号:

Q816

董颖, 马孟丹, 黄卫人. CRISPR-Cas系统的小型化研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-068.

Ying DONG, Mengdan MA, Weiren HUANG. Progress in the miniaturization of CRISPR-Cas systems[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-068.

图/表 8

参考文献 87

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	Cas9	Cas12	Cas13
靶标类型	DNA	DNA	RNA
切割模式	产生平末端的DNA双链断裂	产生粘性末端的DNA双链断裂（部分产生单链DNA切割断裂）	产生单链RNA断裂
PAM序列（用于识别并结合目标）	通常需要一个特定的PAM序列	需要PAM序列（不同于Cas9）	不需要PAM序列，直接靶向于RNA
优势	广泛应用，工具资源丰富，操作简单	因其需要更小的PAM及其粘性末端而更容易进行编辑	针对RNA的编辑能力更强
适用场景	基因编辑及调控、染色质成像；DNA剪切、修复和替换；转录调控^[5]	基因编辑及调控、染色质成像；特异性即时检测^[6]	RNA编辑、调控翻译水平、RNA修饰；基因沉默；抑制病毒RNA复制^[7]
实际应用案例	输血依赖性β地中海贫血（TDT）和镰状细胞病（SCD）^[8]	检测COVID-19^[9]、SARS-Cov-2	开发SHERLOCK技术，检测寨卡和登革热病毒的特定菌株^[10]

	Cas9	Cas12	Cas13
靶标类型	DNA	DNA	RNA
切割模式	产生平末端的DNA双链断裂	产生粘性末端的DNA双链断裂（部分产生单链DNA切割断裂）	产生单链RNA断裂
PAM序列（用于识别并结合目标）	通常需要一个特定的PAM序列	需要PAM序列（不同于Cas9）	不需要PAM序列，直接靶向于RNA
优势	广泛应用，工具资源丰富，操作简单	因其需要更小的PAM及其粘性末端而更容易进行编辑	针对RNA的编辑能力更强
适用场景	基因编辑及调控、染色质成像；DNA剪切、修复和替换；转录调控^[5]	基因编辑及调控、染色质成像；特异性即时检测^[6]	RNA编辑、调控翻译水平、RNA修饰；基因沉默；抑制病毒RNA复制^[7]
实际应用案例	输血依赖性β地中海贫血（TDT）和镰状细胞病（SCD）^[8]	检测COVID-19^[9]、SARS-Cov-2	开发SHERLOCK技术，检测寨卡和登革热病毒的特定菌株^[10]

蛋白	分子量（kDa）	PAM	spacer（nt）	tracrRNA （nt）	直接重复序列（DR）	切割dsDNA	切割ssDNA	实验细胞/细菌
SpCas9^[24]	160	5′-NGG-3′	42	Y	36	Y	N	HEK293T
NmCas9^[25]	162.4	5′-N4GAYW/N4GYTT-3′或 5′-N4GTCT-3′	24	Y	24	Y	N	Mycobacterium smegmatis
SaCas9^[26-27]	126	5′-NNGRRT-3′	21-23	Y	19-24	Y	N	Staphylococcus aureus
CjCas9^[28]	108	5′-N4RYAC-3′	22	Y	35	Y	N	Campylobacter jejuni
mini-SaCas9^[29]	100	5′-NNGRRT-3′	20	N	24	N	N	Staphylococcus aureus
Nme2Cas9^[30]	160	5′-N4CC-3′	22-24	Y	24	Y	N	Neisseria meningitidis
SauriCas9^[31]	118	5′-NNGG-3′	20	Y	36	Y	N	Staphylococcus auricularis
BlatCas9^[32]	120	5′-N4CNAA-3′	17-24	Y	24	Y	N	HEK9T
MISER Cas9^[33]	100	5′-NNRGAA-3′	42	Y	36	N	N	Streptococcus pyogenes
SchCas9^[34]	115	5′-NNGR-3′	21	Y	32	Y	N	/
Nsp2Cas9^[35]	117	5′-N4C-3′	22-26	N	23	Y	N	/
IscB^[36-37]	54	5′-NWRRNA-3′	20	Y	206	Y	N	/

蛋白	分子量（kDa）	PAM	spacer（nt）	tracrRNA （nt）	直接重复序列（DR）	切割dsDNA	切割ssDNA	实验细胞/细菌
SpCas9^[24]	160	5′-NGG-3′	42	Y	36	Y	N	HEK293T
NmCas9^[25]	162.4	5′-N4GAYW/N4GYTT-3′或 5′-N4GTCT-3′	24	Y	24	Y	N	Mycobacterium smegmatis
SaCas9^[26-27]	126	5′-NNGRRT-3′	21-23	Y	19-24	Y	N	Staphylococcus aureus
CjCas9^[28]	108	5′-N4RYAC-3′	22	Y	35	Y	N	Campylobacter jejuni
mini-SaCas9^[29]	100	5′-NNGRRT-3′	20	N	24	N	N	Staphylococcus aureus
Nme2Cas9^[30]	160	5′-N4CC-3′	22-24	Y	24	Y	N	Neisseria meningitidis
SauriCas9^[31]	118	5′-NNGG-3′	20	Y	36	Y	N	Staphylococcus auricularis
BlatCas9^[32]	120	5′-N4CNAA-3′	17-24	Y	24	Y	N	HEK9T
MISER Cas9^[33]	100	5′-NNRGAA-3′	42	Y	36	N	N	Streptococcus pyogenes
SchCas9^[34]	115	5′-NNGR-3′	21	Y	32	Y	N	/
Nsp2Cas9^[35]	117	5′-N4C-3′	22-26	N	23	Y	N	/
IscB^[36-37]	54	5′-NWRRNA-3′	20	Y	206	Y	N	/

蛋白	分子量（kDa）	PAM	spacer（nt）	tracrRNA（nt）	直接重复序列（DR）	切割dsDNA	切割ssDNA	实验细胞/细菌
Cas12d（CasY）^[47]	132	5′-TA-3′	17	Y	26	Y	-	uncultivated microbes
Cas12e5（CasX）^[47-48]	108	5′-TTCN-3′	20	Y	23	Y	-	uncultivated microbes
FnCpf1（Cas12a）^[49]	147	5′--KYTV-3′	20	NA	14	Y	Y	/
AaCas12b（C2c1）^[50]	125	5′-TTN-3′	20	Y	37	Y	Y	Alicyclobacillus acidiphilus
Cas12c（C2c3）^[51-52]	133.09-146.3	5′-TG-3′或5′-TN-3′	17-18	Y	17	Y	Y	HEK293T
Cas12g^[51]	79.2- 91.3	5′-NA-3′	30	Y	36	N	Y	HEK293T
Cas12h^[51]	95.7- 101.64	5′-RTR-3′	-	N	-	Y	Y	HEK293T
Cas12i^[51]	113- 120	5′-TTN-3′	28	N	24	Y	Y	HEK293T
Cas12j^[53-54]	70-80	5′-TBN-3′或5′-TTN-3′	14-20	N	26	Y	Y	HEK293T
CasMINI^[55]	58	5′-TTTR-3′	23	Y	26	Y	Y	TRE3G-GFP HEK293T
UnCas12f^[56-57]	44-77	5′-TTTA-3′	34-39	Y	37	Y	Y	/