Automated Forward Genetic Analysis of Adaptive Immunity

PROJECT SUMMARY
During the past four years, we have made outstanding progress in mutagenizing the mouse germline genome
while keeping immunity under close surveillance. Now four years into the five-year project, we have thoroughly
examined viable hypomorphic mutations in more than half of all protein-encoding genes. We have declared with
high confidence that 1,298 mutations in 638 genes are causative of phenotypes in FACS and/or antibody
response screens. Many of these genes were novel in that their necessity for immune function had been
unknown, and were re-targeted by CRISPR/Cas9 to verify causation. 154 of the 638 candidate genes (24%;
almost all of them novel) were either knocked out or modified with the ENU allele using CRISPR/Cas9 editing,
expanded into pedigrees, and retested for causation of phenotypes detected in screening and automated
mapping. 148 of the 154 genes (96%) were verified as the source of phenotype(s) declared. An additional 156
CRISPR/Cas9 projects are active at this time, some close to completion. Some of our discoveries have been
published; many more are works in progress. However, all results of FACS assays on all mutations, whether
declared causative of phenotype or not, have been de-restricted for public viewing on Mutagenetix, together with
tools that enable search, examination of the original phenotypes and meiotic mapping data, filtering by P-value,
and direction and magnitude of individual phenotypic effects. This will enable other laboratories to pursue
mechanisms of immunological phenotypes alongside us. Knowledge of genes with non-redundant function in
the development and activation of adaptive immune responses is fundamental to immunology and we plan to
pursue screening further. We also plan deeper studies of the mechanism(s) behind phenotypes of particular
interest. Of the phenotypes named for study in our earlier proposal, we have come to understand those caused
by mutations in Trp53bp1, Ampd3, Rnps1, Prkd2, and Snrnp40, and have published papers describing
mechanism. Additional phenotypes (not named in the original proposal) caused by mutations in Rabl3, Gpr89,
Pdia6, Ncstn, Lmbr1l, Stk4, Pacs1, Wdr37, and Mfsd1 have also been elucidated and published or submitted
for publication. We now propose to examine newly verified phenotypes, all the while creating novel phenotypes
for study by ourselves and others. Our work is now guided by a tool (Similarity Heatmap) that measures
relatedness of phenotypes. In flow cytometry screening, a minimum of 34 measurements are made from each
mouse. The results constitute a phenotypic “fingerprint” amenable to tests of statistical similarity. Mutations in
some genes yield results very similar to mutations in other genes, and we can sometimes infer that multiple
genes operate within a single complex of proteins or enzymatic pathway. We have also written software
(Candidate Explorer) to evaluate phenotypes in advance of declaring causation, telling us the likelihood of
validation should we attempt to re-target any gene in question. We restrict our efforts to the most likely novel
candidates, confident that all true causative relationships will ultimately manifest as saturation advances.

Grant Number: 5R01AI125581-10
NIH Institute/Center: NIH
Principal Investigator: BRUCE BEUTLER