Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• Chinese hamster ovary (CHO) cells are crucial hosts for biopharmaceutical protein production.
• Genome engineering of CHO cells is vital for enhancing product quality and yield.

Purpose of the Study:

• To demonstrate the efficacy of CRISPR Cas9 technology for genome editing in CHO cells.
• To develop a bioinformatics tool for identifying CRISPR targets in the CHO-K1 genome.

Main Methods:

• Site-specific gene disruptions were generated in COSMC and FUT8 using CRISPR Cas9 and single guide RNAs (sgRNAs).
• Lectin selection was employed to enhance indel frequency.
• Deep sequencing was used to analyze indel mutations.
• A bioinformatics tool, CRISPy, was developed for sgRNA target identification.

Main Results:

• CRISPR Cas9 achieved high indel frequencies, up to 47.3% in COSMC and 99.7% in FUT8.
• 85% of indels resulted in frameshift mutations, with a preference for single base indels.
• The CRISPy tool identified millions of CRISPR targets and off-target sites in the CHO-K1 genome.

Conclusions:

• CRISPR Cas9 is a robust and efficient genome-editing tool for CHO cells.
• The CRISPy database accelerates genome editing and synthetic biology in CHO cell applications.
• This work facilitates improved biopharmaceutical production using engineered CHO cells.