How to Generate SUCLA2 Knockout in HEK293 Cells Using CRISPR-Cas9

Introduction

SUCLA2 (Succinate-CoA Ligase ADP-Forming Subunit Beta) is a mitochondrial enzyme critical for ATP production, and its dysfunction is linked to mitochondrial DNA depletion syndromes (MDDS) and methylmalonic aciduria. Generating a SUCLA2 knockout (KO) in HEK293 cells provides a valuable model for studying metabolic disorders.

This guide outlines a step-by-step CRISPR-Cas9 protocol to create a stable SUCLA2-KO HEK293 cell line, including design, transfection, validation, and troubleshooting.

Materials Required

1. CRISPR-Cas9 Components

• sgRNA targeting SUCLA2 (designed using CHOPCHOP or CRISPR Design Tool)

• Cas9 plasmid or ribonucleoprotein (RNP) complex (e.g., Alt-R® CRISPR-Cas9 from IDT)

• HEK293 cells (ATCC® CRL-1573™)

• Lipofectamine 3000 (Thermo Fisher) or electroporation system (Neon/Nucleofector)

2. Cell Culture Reagents

• DMEM + 10% FBS + 1% Pen/Strep

• Puromycin (for selection, if using a Cas9-PuroR plasmid)

• Trypsin-EDTA & PBS

3. Validation Tools

• PCR primers for genotyping

• Western blot antibodies (anti-SUCLA2, anti-β-actin control)

• Sanger sequencing/TIDE analysis

Step-by-Step Protocol

1. Design SUCLA2-Specific sgRNA

• Identify exonic regions of SUCLA2 (ENSG00000136197) using NCBI Gene.

• Use CHOPCHOP to select high-efficiency sgRNAs (avoid off-targets with COSMID).

• Example sgRNA sequence (human SUCLA2):

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  5′-GACGUGAUCGAGUCCUCAUG-3′ (Targets Exon 3)  

2. Deliver CRISPR-Cas9 to HEK293 Cells

Option A: Plasmid Transfection

1. Seed HEK293 cells in a 6-well plate (70% confluency).

2. Co-transfect sgRNA+Cas9 plasmid (1 µg each) using Lipofectamine 3000.

3. Add puromycin (1–2 µg/mL) 48h post-transfection for selection.

Option B: RNP Electroporation (Higher Efficiency)

1. Mix Alt-R Cas9 nuclease + sgRNA (IDT) in a 1:1 molar ratio.

2. Electroporate using Neon Transfection System (1100V, 20ms, 2 pulses).

3. Isolate Clonal Populations

• Dilute cells to 0.5 cells/well in a 96-well plate for monoclonal expansion.

• Screen colonies via PCR (primers flanking the target site) and Sanger sequencing.

4. Validate Knockout

• Genomic DNA PCR: Confirm indels via gel electrophoresis (expected ~10–50 bp deletion).

• Western Blot: Use anti-SUCLA2 antibody (Abcam ab129069) to confirm protein loss.

• Functional Assay: Measure mtDNA levels (qPCR) or succinyl-CoA synthetase activity.

  Troubleshooting

  Issue	Solution
  Low knockout efficiency	Optimize sgRNA or switch to RNP delivery
  Off-target effects	Use two sgRNAs or base-edited Cas9 (e.g., BE4max)
  No viable clones	Reduce puromycin dose or use FACS sorting for GFP+ cells

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  Applications of SUCLA2-KO HEK293 Cells

  • Study mitochondrial metabolism defects.

  • Model Leigh syndrome or methylmalonic aciduria.

  • Test therapeutic compounds for mtDNA disorders.

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  Conclusion

  This protocol enables efficient SUCLA2 knockout in HEK293 cells for mitochondrial disease research. For pre-made KO cells, consider vendors like Horizon Discovery or Synthego’s custom services.