A Cost-Effective CRISPR/Cas9-Based Method for Sequencing the Functional Antibody Kappa Chain cDNA from Hybridomas Expressing Aberrant Kappa Chain mRNAs

ABSTRACT One prerequisite for generating recombinant antibodies for therapeutic and non-therapeutic purposes from hybridoma clones is the reliable, efficient, and cost-effective sequencing of the hybridoma clone that produces the desired antibody. However, many hybridoma fusion partners produce aberrant endogenous mRNA transcripts most of which resemble kappa chains. These aberrant kappa chain mRNAs can interfere with or even prevent the determination of the functional murine antibody kappa chain cDNA sequences during the PCR amplification step. In this paper, we report the development of a rapid and cost-effective CRISPR/Cas9 based method to eliminate the aberrant endogenous sequence. We have demonstrated the effectiveness of this method by significantly reducing the number of an endogenous aberrant kappa chain transcript known as the aberrant SP2/0 kappa chain. This transcript is produced by the commonly used fusion partner known as SP2/0 which is a myeloma-derived cell line. Here, we first cloned and sequenced two hybridoma clones using this method (clones A1E7 and A9E11). Our results showed a 24 to 25 percent reduction of the aberrant chain sequences (method 1) from the sequencing results. We then optimized this method (method 2) and used it to clone and sequence a third hybridoma clone (clone 262). This optimized method allowed us to achieve an 88% reduction of the aberrant kappa chain sequences.