Alpha 2.0
Login Register
» Articles » PMID: 26892225

Improved Delivery of Cas9 Protein/gRNA Complexes Using Lipofectamine CRISPRMAX

Overview
Journal Biotechnol Lett
Specialties Biochemistry
Biotechnology
Date 2016 Feb 20
PMID 26892225
Citations 74
Authors
Xin Yu
Xiquan Liang
Huimin Xie
Shantanu Kumar
Namritha Ravinder
Jason Potter
Xavier de Mollerat du Jeu
Jonathan D Chesnut
Affiliations
Soon will be listed here.
Abstract

Objectives: To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.

Results: Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.

Conclusion: Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.

Citing Articles

CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.

Gu B, Li M, Li D, Huang K J Cardiovasc Transl Res. 2025; .

PMID: 39804565 DOI: 10.1007/s12265-024-10587-7.

Efficient mRNA Delivery In Vitro and In Vivo Using a Polycharged Biodegradable Nanomaterial.

Yang X, Xiao J, Staveness D, Zang X Int J Mol Sci. 2025; 25(24.

PMID: 39769382 PMC: 11728123. DOI: 10.3390/ijms252413620.

Highly efficient CRISPR-mediated genome editing through microfluidic droplet cell mechanoporation.

Kim Y, Yun D, Lee J, Jung C, Chung A Nat Commun. 2024; 15(1):8099.

PMID: 39284842 PMC: 11405868. DOI: 10.1038/s41467-024-52493-1.

Universal CAR 2.0 to overcome current limitations in CAR therapy.

Schlegel L, Werbrouck C, Boettcher M, Schlegel P Front Immunol. 2024; 15:1383894.

PMID: 38962014 PMC: 11219820. DOI: 10.3389/fimmu.2024.1383894.

Current Advances in the Functional Genes of Edible and Medicinal Fungi: Research Techniques, Functional Analysis, and Prospects.

Li W, Zou G, Bao D, Wu Y J Fungi (Basel). 2024; 10(5).

PMID: 38786666 PMC: 11121823. DOI: 10.3390/jof10050311.

References
1.
Erazo-Oliveras A, Najjar K, Dayani L, Wang T, Johnson G, Pellois J . Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods. 2014; 11(8):861-7. PMC: 4131206. DOI: 10.1038/nmeth.2998. View
2.
Chu V, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K . Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015; 33(5):543-8. DOI: 10.1038/nbt.3198. View
3.
Copolovici D, Langel K, Eriste E, Langel U . Cell-penetrating peptides: design, synthesis, and applications. ACS Nano. 2014; 8(3):1972-94. DOI: 10.1021/nn4057269. View
4.
Jo J, Hong S, Choi W, Lee D . Cell-penetrating peptide (CPP)-conjugated proteins is an efficient tool for manipulation of human mesenchymal stromal cells. Sci Rep. 2014; 4:4378. PMC: 3953728. DOI: 10.1038/srep04378. View
5.
Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M . Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015; 208:44-53. DOI: 10.1016/j.jbiotec.2015.04.024. View