RNP法基因敲除是一种在各种物种中生成基因敲除的强大工具[1]。CRISPR/Cas9系统已经成为基因组编辑的首选工具，而通过RNP介导的Cas9和sgRNA传递在原始T细胞中已被证明具有高效率[1]。下面，我们将讨论关于RNP介导的基因组编辑的文献，包括其优势、局限性和应用。

RNP介导的基因组编辑的优势：

高效率：RNP介导的Cas9和sgRNA传递在原始T细胞中已被证明具有高效率[1]。这种方法在人群水平上导致了目标基因表达的几乎完全丧失，减轻了选择的需求[1]。

减少非特异性效应：与质粒传递相比，RNP介导的Cas9和sgRNA传递已被证明减少了非特异性效应[2]。

减少细胞毒性：Cas9蛋白对细胞的毒性较质粒传递较小[2]。

快速高效：RNP介导的Cas9和sgRNA传递是一种快速高效的基因编辑方法[2]。

RNP介导的基因组编辑的局限性：

载体容量有限：Cas9蛋白和sgRNA的大小限制了可以编辑的目标序列的大小[2]。

传递方法有限：RNP介导的Cas9和sgRNA传递需要专门的传递方法，如电穿孔或核穿孔[2]。

编辑窗口有限：RNP介导的Cas9和sgRNA传递在传递后通常有限的编辑窗口，通常为24-48小时[2]。

RNP介导的基因组编辑的应用：

目标基因的发现和验证：RNP介导的Cas9和sgRNA传递可用于研究原始T细胞中的基因功能[1]。这种方法极大地扩展了原始T细胞中目标基因的发现和验证的可行性，并简化了下一代免疫治疗的基因编辑过程[1]。

血液造血细胞的编辑：RNP介导的Cas9和sgRNA传递在造血细胞系和原代细胞中已被证明具有高效率[2]。这种方法已被用于在急性髓系白血病（AML）细胞系中诱导普遍的造血标记CD45的敲除，以及在B细胞癌细胞系中敲除两个B细胞标记物（CD19和CD22）[2]。

总之，RNP介导的基因组编辑是一种在各种物种中生成基因敲除的强大工具。这种方法具有高效率、减少非特异性效应、减少细胞毒性和快速高效的优点。然而，它也存在一些局限性，包括载体容量有限、传递方法有限和编辑窗口有限。RNP介导的基因组编辑具有多种应用，包括原始T细胞中目标基因的发现和验证以及血液造血细胞的编辑。

Citations:

[1] Seki, A., & Rutz, S. (2018). Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. Journal of Experimental Medicine, 215(3), 985-997.

[2] Gundry, M. C., Brunetti, L., Wagner, D. L., Hsu, J., Velasquez, M. P., Gottschalk, S., ... & Goodell, M. (2016). Fast and Efficient Gene Editing in Human Hematopoietic Cells. Blood, 128(22),4704.https://www.semanticscholar.org/paper/7066a91b374475979104a1a6d63eba3c1c478b43

Ribonucleoprotein (RNP)-mediated genome editing is a powerful tool for generating gene knockouts across a variety of species[1]. The CRISPR/Cas9 system has become the tool of choice for genome editing, and RNP-mediated delivery of Cas9 and sgRNA has been shown to be highly efficient in primary T cells[1]. Here, we will discuss the literature on RNP-mediated genome editing, including its advantages, limitations, and applications.

Advantages of RNP-mediated genome editing:

- High efficiency: RNP-mediated delivery of Cas9 and sgRNA has been shown to be highly efficient in primary T cells[1]. This method results in near complete loss of target gene expression at the population level, mitigating the need for selection[1].

- Reduced off-target effects: RNP-mediated delivery of Cas9 and sgRNA has been shown to reduce off-target effects compared to plasmid-based delivery[2].

- Reduced cellular toxicity: Cas9 protein is less toxic to cells than plasmid-based delivery[2].

- Rapid and efficient: RNP-mediated delivery of Cas9 and sgRNA is a rapid and efficient method for gene editing[2].

Limitations of RNP-mediated genome editing:

- Limited cargo capacity: The size of the Cas9 protein and sgRNA limits the size of the target sequence that can be edited[2].

- Limited delivery methods: RNP-mediated delivery of Cas9 and sgRNA requires specialized delivery methods, such as electroporation or nucleofection[2].

- Limited editing window: RNP-mediated delivery of Cas9 and sgRNA has a limited editing window, typically 24-48 hours after delivery[2].

Applications of RNP-mediated genome editing:

- Target gene discovery and validation: RNP-mediated delivery of Cas9 and sgRNA can be used to study gene function in primary T cells[1]. This method greatly extends the feasibility of target gene discovery and validation in primary T cells and simplifies the gene editing process for next-generation immunotherapies[1].

- Hematopoietic cell editing: RNP-mediated delivery of Cas9 and sgRNA has been shown to be highly efficient in hematopoietic cell lines and primary cells[2]. This method has been used to induce knockout of the ubiquitous hematopoietic marker CD45 in acute myeloid leukemia (AML) cell lines, as well as knockout of two B-cell markers (CD19 and CD22) in B-cell cancer cell lines[2].

In conclusion, RNP-mediated genome editing is a powerful tool for generating gene knockouts in a variety of species. This method has several advantages, including high efficiency, reduced off-target effects, reduced cellular toxicity, and rapid and efficient delivery. However, it also has limitations, including limited cargo capacity, limited delivery methods, and a limited editing window. RNP-mediated genome editing has several applications, including target gene discovery and validation in primary T cells and hematopoietic cell editing.

Citations:

[1] Seki, A., & Rutz, S. (2018). Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. Journal of Experimental Medicine, 215(3), 985-997.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839763/

[2] Gundry, M. C., Brunetti, L., Wagner, D. L., Hsu, J., Velasquez, M. P., Gottschalk, S., ... & Goodell, M. (2016). Fast and Efficient Gene Editing in Human Hematopoietic Cells. Blood, 128(22), 4704.https://www.semanticscholar.org/paper/7066a91b374475979104a1a6d63eba3c1c478b43