After lung vaccination with riboflavin metabolizing live bacteria, Mucosal-associated invariant T (MAIT) cells increase in number and remained increased in the tissue long-term after bacterial clearance. Some of the transcriptional changes observable long-term after exposure are also found in canonical T cell memory populations when compared to naïve cells. We compared the gene expression programs of conventional immunological memory to genes altered in these long-term MAIT cell subpopulations. Overlapping and MAIT-exclusive gene hits were used to perform a pooled in vivo CRISPR screen against 786 gene targets. Expanded MAIT cells were sorted from the lungs of Cas9 transgenic mice 6 days after vaccination with Salmonella BRD509 strain and transduced with a pooled of MSCV retrovirus expressing 2776 different guide RNAs. Transduced cells were then injected into congenic marked (Ly5.1+), Rag-2 KO mice. MAIT cells were collected from these mice 2 weeks after injection (day 0), or 6 days and 40 days after re-challenge with Salmonella BRD509. MAIT cells at these timepoints were sequenced to analyze relative abundance of single guide RNA (sgRNA) at this timepoint. We found that most hits were depleted at day 6, thus important for the activation and proliferation of MAIT cell . Targets depleted selectively at day 40 were indicative of genes important for long-term maintenance of memory-like MAIT cells. However, Syndecan-1 sgRNAs were over-represented at day 40. Syndecan-1 (Sdc1) is a heparan sulfate proteoglycan receptor shown to negatively regulate the production of Interleukin-17 (IL-17) by CD4 Th17 T cells, but also by innate-like T cells, as Tγδ17 cells and natural killer T (NKT)17 cells. Many mouse MAIT cells produce IL-17 (MAIT17 cells), and our data suggest that Syndecan-1 negatively regulates this population as well. Following other hits, this in vivo approach will allow us to determine essential genes driving the generation of long-term MAIT cells that provide defense against infections and other useful immune responses.