CRISPR/Cas9及其衍生编辑器在衰老研究中的应用进展

doi:10.12211/2096-8280.2021-100

摘要/Abstract

摘要：

衰老逐渐成为威胁人类健康的重要因素，其中基因突变及其累积是引发衰老的因素之一。基因编辑技术可以纠正或清除错误基因突变，从而具有延缓衰老和治疗衰老相关疾病的潜力。CRISPR/Cas（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein）的基因编辑工具是基于细菌和古细菌获得性免疫系统发明的，已证明可修改多种生物体的基因组。通过对Cas蛋白的定点突变和表达优化改造，可提高CRISPR/Cas系统在靶向位点的编辑效率、保真性及降低其脱靶效应。随后，科学家们发明了许多基于Cas蛋白的编辑器，如碱基编器、引导编辑器、转座/重组酶等。CRISPR/Cas及其衍生的编辑器，可实现多种形式精确的基因修饰，从而满足不同的基因编辑需求。本文主要概述了CRISPR/Cas系统及其种类、Cas9（CRISPR-associated protein 9）蛋白及其变体、基于Cas9蛋白衍生的基因编辑器，并讨论了这些编辑器在衰老及其相关疾病，如早衰综合症、心血管疾病、年龄相关性黄斑变性、神经退行性疾病等方面的应用研究进展。未来，高精确基因编辑器和递送技术的发明，将加速基因编辑技术用于治疗衰老相关疾病和逆转衰老的基础与临床研究，最终实现人类健康衰老。

关键词: CRISPR/Cas系统, Cas蛋白衍生编辑器, 衰老及衰老相关疾病

Abstract:

Aging is a complicated process, with aging individuals exhibiting a decline in organ functions and the development of multiple diseases, making it one of major threats to human health. Senescence is often caused by cellular degeneration, which is characterized by morphological and metabolic changes, chromatin remodeling, altered gene expression and a pro-inflammatory phenotype called senescence-associated secretory phenotype (SASP). Moreover, gene mutations and their accumulation are one of the factors triggering aging, and gene editing technology can thus be used to correct or remove erroneous gene mutations, thereby retarding cellular senescence and individual aging as well as treating individual aging-related diseases. Discovered based on the acquired immune system of bacteria and archaea, CRISPR/Cas was initially used to knock out genes, and then its application has been extended to modify the genomes of various organisms. Amino acid mutation and protein structure optimization of Cas protein can not only expand the range of CRISPR/Cas targeted sequence, but also improve its editing efficiency and fidelity to reduce off-target effect. Many Cas protein-based editors, such as base editors, prime editors, transposases/recombinases, have also been invented, which can achieve single-nucleotide editing and integration of large fragments. CRISPR/Cas and its derived editors enable precise gene modifications to meet requirement for editing different genes. In this article, we summarize the CRISPR/Cas system and its types, Cas9 protein and its variants, gene editors derived from Cas9 protein, and discuss their potential applications in treating aging and aging related diseases including progeria syndrome, cardiovascular disease, age-related macular degeneration and neurodegenerative diseases. In the future, the development of gene editing tools with highly targeted editing and low-level off-target and the optimization of delivery methods will accelerate the transfer of the gene editing technology to research and clinical application. Not only can gene editing be used to treat diseases caused by gene mutations, but also become a favorable tool for the treatment of aging and aging related diseases.

Key words: CRISPR/ Cas9, Cas protein derived editors, aging and age-related diseases

中图分类号:

龚仕涛, 王宇, 陈宇庭. CRISPR/Cas9及其衍生编辑器在衰老研究中的应用进展[J]. 合成生物学, 2022, 3(1): 66-77.

Shitao GONG, Yu WANG, Yuting CHEN. Advances in application of CRISPR/Cas9 and its derivative editors in aging research[J]. Synthetic Biology Journal, 2022, 3(1): 66-77.

图/表 3

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Name	Description of protein variant or mutations	PAM (5′ to 3′)
SpCas9	Native Streptococcus pyogenes Cas9	NGG^[28]
VRER SpCas9	D1135V, G1218R, R1335E, T1337R	NGCG^[29]
VQR SpCas9	D1135V, R1335Q, T1337R	NGAN or NGNG^[29]
EQR SpCas9	D1135E, R1335Q, T1337R	NGAG^[29]
xCas9-3.7	A262T, R324L, S409I, E480K, E543D, M694I, E1219V	NG, GAA, GAT^[30]
eSpCas9 (1.0)	K810A, K1003A, R1060A	NGG
eSpCas9 (1.1)	K848A, K1003A, R1060A	NGG
Cas9-HF1	N497A, R661A, Q695A, Q926A	NGG
HypaCas9	N692A, M694A, Q695A, H698A	NGG
evoCas9	M495V, Y515N, K526E, R661Q	NGG
HiFi Cas9	R691A	NGG
ScCas9	Native Streptococcus canis Cas9	NNG^[31]
StCas9	Native Streptococcus thermophilus Cas9	NNAGAAW^[32-33]
NmCas9	Native Neisseria meningitidis Cas9	NNNNGATT^[34-36]
SaCas9	Native Staphylococcus aureus Cas9	NNGRRT^[37]
CjCas9	Native Campylobacter jejuni Cas9	NNNVRYM^[38]
CasX	Phyla Deltaproteobacteria and Planctomycetes	TTCN^[39]
SpG	variants of Streptococcus pyogenes Cas9	NGN^[40]
SpRY	SpCas9 variant	NHN^[40]
SpCas9	R1333K, R1335 and T1337N	NRRH, NRCH and NRTH^[41]
SpCas9	computational models	NNNN^[42]

Name	Description of protein variant or mutations	PAM (5′ to 3′)
SpCas9	Native Streptococcus pyogenes Cas9	NGG^[28]
VRER SpCas9	D1135V, G1218R, R1335E, T1337R	NGCG^[29]
VQR SpCas9	D1135V, R1335Q, T1337R	NGAN or NGNG^[29]
EQR SpCas9	D1135E, R1335Q, T1337R	NGAG^[29]
xCas9-3.7	A262T, R324L, S409I, E480K, E543D, M694I, E1219V	NG, GAA, GAT^[30]
eSpCas9 (1.0)	K810A, K1003A, R1060A	NGG
eSpCas9 (1.1)	K848A, K1003A, R1060A	NGG
Cas9-HF1	N497A, R661A, Q695A, Q926A	NGG
HypaCas9	N692A, M694A, Q695A, H698A	NGG
evoCas9	M495V, Y515N, K526E, R661Q	NGG
HiFi Cas9	R691A	NGG
ScCas9	Native Streptococcus canis Cas9	NNG^[31]
StCas9	Native Streptococcus thermophilus Cas9	NNAGAAW^[32-33]
NmCas9	Native Neisseria meningitidis Cas9	NNNNGATT^[34-36]
SaCas9	Native Staphylococcus aureus Cas9	NNGRRT^[37]
CjCas9	Native Campylobacter jejuni Cas9	NNNVRYM^[38]
CasX	Phyla Deltaproteobacteria and Planctomycetes	TTCN^[39]
SpG	variants of Streptococcus pyogenes Cas9	NGN^[40]
SpRY	SpCas9 variant	NHN^[40]
SpCas9	R1333K, R1335 and T1337N	NRRH, NRCH and NRTH^[41]
SpCas9	computational models	NNNN^[42]