8.9
CRISPR/Cas9 基因组编辑
CRISPR-Cas系统作为细菌防御病毒和质粒等外源入侵遗传物质的机制，构成了其作为强大基因组编辑工具的基础。该系统最初在原核生物中发现，现已被改造用于多种生物的基因工程革命，包括植物、动物及人类。其核心组分Cas9是一种来源于化脓性链球菌（Streptococcus pyogenes）的核酸内切酶…
CRISPR-Cas 系统可保护细菌免受外源遗传元件的侵害。
在实验室中，利用 Cas-9 编程该系统以对植物、动物和人类的基因进行编辑。
Cas9 是一种来自 Streptococcus pyogenes 的内切核酸酶，通过与合成的向导 RNA（sgRNA）共同递送进入细胞。
sgRNA 将 Cas9 引导至原间隔序列邻近基序序列，使其能够结合并在靶位点切割 DNA。
对于基因插入，Cas9 在一条 sgRNA 的引导下切割靶位点，从而通过同源重组插入新基因。
进行基因敲除时，两条sgRNA引导Cas9在目标区域的两端进行切割。
基因被切除，修复系统将断裂的末端连接起来。
CRISPR-Cas9 已被设计用于通过切除感染细胞中的 HIV DNA 来使其失活。像 Csy4 这样的工程化系统可靶向游离的 HIV RNA，但这些方法仍处于实验阶段。
除了靶向单个位点外，CRISPR-Cas9 还可以同时编辑多个基因，例如清除多个逆转录病毒拷贝。
View the full transcript and gain access to JoVE Core videos
Q1: How does Cas9 identify and cut DNA at the target site?
Cas9, an endonuclease from Streptococcus pyogenes, uses a synthetic guide RNA (sgRNA) to locate specific DNA sequences. The sgRNA directs Cas9 to the Protospacer Adjacent Motif (PAM) sequence, typically 5'-NGG-3', allowing Cas9 to bind and introduce a double-stranded break at the precise target location.
Q2: What is the difference between gene insertion and gene deletion using CRISPR-Cas9?
Gene insertion uses one sgRNA to guide Cas9 to cleave a site, then homologous recombination inserts new genetic material. Gene deletion employs two sgRNAs directing Cas9 to cut both ends of a target region, excising the DNA fragment, which the cell's repair system then rejoins through non-homologous end joining.
Q3: How can CRISPR-Cas9 be used to treat HIV infections?
CRISPR-Cas9 has been designed to inactivate HIV by excising integrated viral DNA from infected cells. Additionally, engineered systems like Csy4 target free HIV RNA to degrade it, though these antiviral strategies remain experimental and represent emerging therapeutic approaches for combating viral infections.
Q4: What is the original function of the CRISPR-Cas system in bacteria?
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids. Originally discovered in prokaryotes, the antiviral system of bacteria and archaea has been repurposed as a powerful genome-editing tool for laboratory applications across diverse organisms.
Q5: Can CRISPR-Cas9 edit multiple genes simultaneously?
Yes, CRISPR-Cas9 enables multiplexed genome editing, allowing simultaneous modification of multiple genes in a single organism. This capability is particularly advantageous in complex genetic studies and therapeutic applications, such as removing multiple retroviral copies from infected cells in one editing event.
Q6: What organisms can be edited using CRISPR-Cas9 technology?
CRISPR-Cas9 has been reprogrammed to modify the genomes of diverse organisms, including plants, animals, and humans. This versatility makes it a revolutionary tool for genetic engineering across a wide range of biological systems and research applications in both basic and applied science.
Q7: How does the synthetic guide RNA direct Cas9 to the correct DNA location?
The synthetic single guide RNA (sgRNA) contains a complementary sequence that guides Cas9 to a specific genomic locus adjacent to the Protospacer Adjacent Motif (PAM). This precise targeting ensures Cas9 binds and cuts only at the intended DNA location, enabling accurate and efficient genome editing with minimal off-target effects.