Figure 1 (Hsu, Lander et al. 2014)
CRISPR/Cas9基因敲除技术简介
CRISPR/Cas9 是进行基因编辑的强大工具，基因敲除是在目的基因的上下游各设计一条向导RNA（向导RNA1，向导RNA2），将其与含有Cas9蛋白编码基因的质粒一同转入细胞中，向导RNA通过碱基互补配对可以靶向PAM附近的目标序列，Cas9蛋白会使该基因上下游的DNA双链断裂。而生物体自身存在着DNA损伤修复的应答机制，会将断裂上下游两端的序列连接起来，从而实现了细胞中目标基因的精确敲除。目的基因待编辑的区域附近存在相对保守的PAM序列（NGG），向导RNA要与PAM上游的序列碱基互补配对(Chakrabarti, Henser-Brownhill et al. 2019)。
CRISPR/Cas9基因敲除技术使用范围
使用CRISPR/Cas9构建敲除细胞系，在这类细胞系中，靶标基因可以精确删除，完全去除其编码的蛋白。
技术优势
1.CRISPR/Cas9提高了基因敲除的准确性和特异性
2.适用于敲除片段3000nt以上的非编码RNA
构建敲除基因的稳转细胞系，后续可用于动物体内实验、细胞表型实验检测等
2.适用于敲除片段3000nt以上的非编码RNA
构建敲除基因的稳转细胞系，后续可用于动物体内实验、细胞表型实验检测等
参考文献
1.Bagaria, J., Y. Moon, E. Bagyinszky, K. H. Shim, S. S. A. An, S. Kim and S. H. Han (2022). ``Whole Exome Sequencing Reveals a Novel APOE Mutation in a Patient With Sporadic Early-Onset Alzheimer's Disease.`` Front Neurol 13: 899644.
2. Chakrabarti, A. M., T. Henser-Brownhill, J. Monserrat, A. R. Poetsch, N. M. Luscombe and P. Scaffidi (2019). ``Target-Specific Precision of CRISPR-Mediated Genome Editing.`` Mol Cell 73(4): 699-713 e696.
3. Chiu, Y. J., H. Y. Weng, Y. Y. Lin, Y. F. Lin, Y. C. Yeh, C. K. Perng, H. Ma, S. F. Tsai and T. Y. Chou (2022). ``Genomic profiling with whole-exome sequencing revealed distinct mutations and novel pathways in Asian melanoma.`` J Dermatol.
4. Horjus, J., T. van Mourik-Banda, M. A. P. Heerings, M. Hakobjan, W. De Witte, D. J. Heersema, A. J. Jansen, E. M. M. Strijbis, B. A. de Jong, A. E. J. Slettenaar, E. Zeinstra, E. L. J. Hoogervorst, B. Franke, W. Kruijer, P. J. Jongen, L. J. Visser and G. Poelmans (2022). ``Whole Exome Sequencing in Multi-Incident Families Identifies Novel Candidate Genes for Multiple Sclerosis.`` Int J Mol Sci 23(19).
5. Majewski, J., J. Schwartzentruber, E. Lalonde, A. Montpetit and N. Jabado (2011). ``What can exome sequencing do for you?`` Journal of medical genetics 48(9): 580-589.
6. Umlai, U. I., D. K. Bangarusamy, X. Estivill and P. V. Jithesh (2022). ``Genome sequencing data analysis for rare disease gene discovery.`` Brief Bioinform 23(1).
7. Xiao, L., D. Wu, Y. Sun, D. Hu, J. Dai, Y. Chen and D. Wang (2022). ``Whole-exome sequencing reveals genetic risks of early-onset sporadic dilated cardiomyopathy in the Chinese Han population.`` Sci China Life Sci 65(4): 770-780.
2. Chakrabarti, A. M., T. Henser-Brownhill, J. Monserrat, A. R. Poetsch, N. M. Luscombe and P. Scaffidi (2019). ``Target-Specific Precision of CRISPR-Mediated Genome Editing.`` Mol Cell 73(4): 699-713 e696.
3. Chiu, Y. J., H. Y. Weng, Y. Y. Lin, Y. F. Lin, Y. C. Yeh, C. K. Perng, H. Ma, S. F. Tsai and T. Y. Chou (2022). ``Genomic profiling with whole-exome sequencing revealed distinct mutations and novel pathways in Asian melanoma.`` J Dermatol.
4. Horjus, J., T. van Mourik-Banda, M. A. P. Heerings, M. Hakobjan, W. De Witte, D. J. Heersema, A. J. Jansen, E. M. M. Strijbis, B. A. de Jong, A. E. J. Slettenaar, E. Zeinstra, E. L. J. Hoogervorst, B. Franke, W. Kruijer, P. J. Jongen, L. J. Visser and G. Poelmans (2022). ``Whole Exome Sequencing in Multi-Incident Families Identifies Novel Candidate Genes for Multiple Sclerosis.`` Int J Mol Sci 23(19).
5. Majewski, J., J. Schwartzentruber, E. Lalonde, A. Montpetit and N. Jabado (2011). ``What can exome sequencing do for you?`` Journal of medical genetics 48(9): 580-589.
6. Umlai, U. I., D. K. Bangarusamy, X. Estivill and P. V. Jithesh (2022). ``Genome sequencing data analysis for rare disease gene discovery.`` Brief Bioinform 23(1).
7. Xiao, L., D. Wu, Y. Sun, D. Hu, J. Dai, Y. Chen and D. Wang (2022). ``Whole-exome sequencing reveals genetic risks of early-onset sporadic dilated cardiomyopathy in the Chinese Han population.`` Sci China Life Sci 65(4): 770-780.
Figure 2 (Van Nostrand, Gelboin-Burkhart et al. 2017)
参考文献：
Hsu, P. D., E. S. Lander and F. Zhang (2014). “Development and applications of CRISPR-Cas9 for genome engineering.” Cell 157(6): 1262-1278.
Van Nostrand, E. L., C. Gelboin-Burkhart, R. Wang, G. A. Pratt, S. M. Blue and G. W. Yeo (2017). “CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins.” Methods 118: 50-59.