CRISPR-Cas9 knockout in cultured mammalian cells
Materials Required
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Principle
CRISPR-Cas9 knockout in cultured mammalian cells uses an sgRNA to direct Cas9 to a complementary genomic sequence adjacent to a compatible PAM; Cas9 creates a targeted DNA double-strand break, and repair by non-homologous end joining can introduce insertions or deletions that disrupt the coding sequence or functional genomic element. The readout of knockout is detection of edited alleles and loss of gene product or phenotype, commonly by PCR/Sanger-sequence trace decomposition, targeted sequencing, immunoblotting, immunostaining, or flow cytometry when the target protein is detectable at the cell surface.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use cultured mammalian cells, complete cell-culture medium appropriate for the cell type, Cas9-sgRNA plasmid, lentiviral or integrase-deficient lentiviral delivery reagents, or purified Cas9 protein plus sgRNA for RNP delivery;
• Published protocols support plasmid, lentiviral, and RNP-based delivery in mammalian cells.
• Use PCR primers flanking the sgRNA target site to amplify edited genomic DNA for mutation detection;
• Sanger sequencing of the PCR product can be analyzed by sequence-trace decomposition to estimate indel frequency.
• Use antibodies only when the target protein has a validated antibody-based readout;
• Published CRISPR knockout evaluation used flow cytometry for CD19 surface loss and Sanger sequencing for genotype confirmation.
• Use standard mammalian cell-culture equipment, a transfection or electroporation system compatible with the chosen delivery format, PCR equipment, Sanger sequencing access, and flow cytometry or immunoblotting equipment when protein-level validation is planned.
• Published protocols support plasmid, lentiviral, and RNP-based delivery in mammalian cells.
• Use PCR primers flanking the sgRNA target site to amplify edited genomic DNA for mutation detection;
• Sanger sequencing of the PCR product can be analyzed by sequence-trace decomposition to estimate indel frequency.
• Use antibodies only when the target protein has a validated antibody-based readout;
• Published CRISPR knockout evaluation used flow cytometry for CD19 surface loss and Sanger sequencing for genotype confirmation.
• Use standard mammalian cell-culture equipment, a transfection or electroporation system compatible with the chosen delivery format, PCR equipment, Sanger sequencing access, and flow cytometry or immunoblotting equipment when protein-level validation is planned.
Experimental Procedure
• Select sgRNAs targeting an early constitutive coding exon when the goal is loss of protein function, and prioritize guides with empirically supported design rules that maximize on-target activity and reduce predicted off-target activity.
• Prepare at least one non-targeting sgRNA negative control and, when feasible, more than one independent sgRNA per target gene so that shared phenotypes can be distinguished from guide-specific effects.
• Seed cells so that they are healthy and actively growing at delivery; the exact cell number, confluence, and delivery condition should be optimized for the cell type because published CRISPR protocols report cell-type-specific transfection, electroporation, or viral transduction workflows rather than a universal parameter.
• Deliver CRISPR components using one literature-supported format: plasmid expression of Cas9 and sgRNA, viral delivery of CRISPR components, or preassembled Cas9-sgRNA RNPs.
• For RNP editing, preassemble purified Cas9 protein with sgRNA before delivery;
• Kim et al. reported delivery of Cas9 RNPs into cultured human cells, including K562 cells, fibroblasts, and pluripotent stem cells, and observed rapid editing with reduced plasmid-associated off-target effects.
• For lentiviral or integrase-deficient lentiviral delivery, produce and apply vectors according to published lentiviral CRISPR protocols, then culture edited cells before genotyping or phenotype testing;
• Integrase-deficient lentiviral vectors were developed to deliver CRISPR components while reducing risks associated with stable vector integration.
• After delivery, allow cells to recover and expand before DNA extraction and phenotype testing;
• Published protocols evaluate editing after sufficient culture time for mutation accumulation and protein loss, but the exact timing depends on target protein stability, delivery format, and cell type.
• Extract genomic DNA, PCR-amplify the target locus, perform Sanger sequencing, and analyze mixed sequence traces with TIDE-style decomposition to estimate indel frequency in the edited cell population.
• Confirm knockout at the protein or functional level when the target gene product can be measured;
• CD19 knockout evaluation used flow cytometry to quantify loss of CD19-positive cells and Sanger sequencing to confirm genomic editing.
• Interpret knockout experiments using non-targeting sgRNA controls, untreated or mock-delivered controls, and independent sgRNAs targeting the same gene;
• Guide activity and off-target profiles vary across sgRNAs, so conclusions should not rely on a single unvalidated guide.
• Prepare at least one non-targeting sgRNA negative control and, when feasible, more than one independent sgRNA per target gene so that shared phenotypes can be distinguished from guide-specific effects.
• Seed cells so that they are healthy and actively growing at delivery; the exact cell number, confluence, and delivery condition should be optimized for the cell type because published CRISPR protocols report cell-type-specific transfection, electroporation, or viral transduction workflows rather than a universal parameter.
• Deliver CRISPR components using one literature-supported format: plasmid expression of Cas9 and sgRNA, viral delivery of CRISPR components, or preassembled Cas9-sgRNA RNPs.
• For RNP editing, preassemble purified Cas9 protein with sgRNA before delivery;
• Kim et al. reported delivery of Cas9 RNPs into cultured human cells, including K562 cells, fibroblasts, and pluripotent stem cells, and observed rapid editing with reduced plasmid-associated off-target effects.
• For lentiviral or integrase-deficient lentiviral delivery, produce and apply vectors according to published lentiviral CRISPR protocols, then culture edited cells before genotyping or phenotype testing;
• Integrase-deficient lentiviral vectors were developed to deliver CRISPR components while reducing risks associated with stable vector integration.
• After delivery, allow cells to recover and expand before DNA extraction and phenotype testing;
• Published protocols evaluate editing after sufficient culture time for mutation accumulation and protein loss, but the exact timing depends on target protein stability, delivery format, and cell type.
• Extract genomic DNA, PCR-amplify the target locus, perform Sanger sequencing, and analyze mixed sequence traces with TIDE-style decomposition to estimate indel frequency in the edited cell population.
• Confirm knockout at the protein or functional level when the target gene product can be measured;
• CD19 knockout evaluation used flow cytometry to quantify loss of CD19-positive cells and Sanger sequencing to confirm genomic editing.
• Interpret knockout experiments using non-targeting sgRNA controls, untreated or mock-delivered controls, and independent sgRNAs targeting the same gene;
• Guide activity and off-target profiles vary across sgRNAs, so conclusions should not rely on a single unvalidated guide.
Troubleshooting
Low or undetectable indel frequency
May be due to weak on-target activity of the selected sgRNARedesign and test alternative sgRNAs using empirically optimized sgRNA design rules, as sgRNA activity varies widely and optimized guide design improves CRISPR-Cas9 editing performance.
Apparent phenotype not reproduced by another sgRNA
May result from guide-specific off-target editing or guide-specific activity differencesUse multiple independent sgRNAs and evaluate predicted off-target risk, as SpCas9 mismatch tolerance depends on mismatch number, position, and distribution.
Genomic editing detected but protein knockout incomplete
May be due to in-frame indels, mixed edited populations, or residual protein persistenceCombine genotype analysis with a protein-level assay such as flow cytometry when the target protein has a measurable surface marker.
Plasmid delivery causing concern about prolonged Cas9 exposure or plasmid-derived DNA effects
May stem from persistent plasmid expressionUse Cas9-sgRNA RNP delivery when compatible with the cell type, as RNP editing was reported to act rapidly and reduce plasmid-associated off-target effects.
References:
- [1]. Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308. [Content Brief]
- [2]. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823. [Content Brief]
- [3]. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826. [Content Brief]
- [4]. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184-191. [Content Brief]
- [5]. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827-832. [Content Brief]
- [6]. Kim S, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24(6):1012-1019. [Content Brief]
- [7]. Vijayraghavan S, et al. A protocol for the production of integrase-deficient lentiviral vectors for CRISPR/Cas9-mediated gene knockout in dividing cells. J Vis Exp. 2017;(130):56915. [Content Brief]
- [8]. Brinkman EK, et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014;42(22):e168. [Content Brief]
- [9]. Inwood SL, et al. Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing. BioTechniques. 2022;72(6):279-286. [Content Brief]
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