CRISPR/Cas9 generated human CD46, CD55 and CD59 knockout cell lines as a tool for complement research.

Area of Science:

• Immunology
• Cell Biology

Background:

• Complement system dysregulation is implicated in diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome.
• Investigating complement regulator deficiencies using patient cells is challenging due to limited access and heterogeneity.
• Existing methods to inhibit complement regulators on healthy cells may introduce confounding antibody-mediated effects.

Purpose of the Study:

• To develop a robust in vitro model for studying complement activation and regulation on human cells.
• To create human cell lines with specific deficiencies in complement regulatory proteins.
• To overcome limitations associated with patient-derived cells and blocking antibodies.

Main Methods:

• CRISPR/Cas9 gene editing was employed to generate knockout (KO) HAP1 cell lines lacking CD46, CD55, and/or CD59.
• Single-cell derived lines were validated using Sanger sequencing and flow cytometry.
• Complement deposition (C3 and C4) was assessed via flow cytometry after exposing cells to normal human serum.

Main Results:

• Single KO cell lines for CD46, CD55, and CD59 were successfully generated, alongside double and triple KO combinations.
• Deletion of CD46 and CD55 increased C3/C4 and C3 deposition, respectively, upon classical pathway activation, confirming their regulatory roles.
• Alternative pathway activation led to C3 deposition only on triple KO cells, indicating resistance to spontaneous activation and a potential role for CD59.

Conclusions:

• The generated complement regulator knockout cell lines serve as valuable tools for in vitro studies of human complement activation.
• These cell lines can aid in evaluating novel therapeutic complement inhibitors.
• The study identified a potential novel role for CD59 in regulating complement component C3 deposition.