High-resolution scRNA-seq reveals genomic determinants of antigen expression hierarchy in African Trypanosomes

Antigenic variation is an immune evasion strategy used by many different pathogens. It involves the periodic, non-random switch in the expression of different antigens throughout an infection. How the observed hierarchy in antigen expression is achieved has remained a mystery. A key challenge in uncovering this process has been the inability to track transcriptome changes and potential genomic rearrangements in individual cells during a switch event. Here, we report the establishment of a highly sensitive single-cell RNA-seq (scRNA-seq) approach for the model protozoan parasite Trypanosoma brucei. This approach has revealed genomic rearrangements that occur in individual cells during a switch event. Our data show that following a double-strand break (DSB) in the transcribed antigen-coding gene - an important trigger for antigen switching - the type of repair mechanism and the resultant antigen expression depend on the availability of a homologous repair template in the genome. When such a template was available, repair proceeded through segmental gene conversion, creating new, mosaic antigen-coding genes. Conversely, in the absence of a suitable template, a telomere-adjacent antigen-coding gene from a different part of the genome was activated by break-induced replication. Our results reveal the critical role of available repair sequence in the antigen selection mechanism. Additionally, our study demonstrates the power of highly sensitive scRNA-seq methods in detecting genomic rearrangements that drive transcriptional changes at the single-cell level.