Cas9 RNP targeting cis-regulatory elements in lymphocytes

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Project Summary CRISPR tools for genome editing have revolutionized and accelerated the functional interrogation of coding genes in model animal systems and in human somatic cells, including primary lymphocytes that play critical roles in the immunopathology of allergy and asthma. We propose to adapt and optimize these tools for miRNA- directed pathway discovery. First, will create pooled retroviral CRISPR libraries for targets that we have empirically identified for miR-29, a miRNA with critical functions in both T cells and B cells. Second, we will extend next generation transfection-based CRISPR systems to target cis-regulatory sequences that do not code for protein, but mediate regulatory interactions between miRNAs and target mRNAs. Success in these aims will enable detailed mechanistic analysis of miRNA:target networks that govern immune responses in asthma and allergy. A second important goal of this proposal is to support the career and scientific development of Celeste Garza, a post-baccalaureate researcher gaining research experience as a junior specialist in the Ansel laboratory. The proposed experiments build on skills she has learned in the Ansel lab, and will provide the opportunity for her to develop a research narrative and scientific achievements as prepares to apply for PhD programs in the coming year.