CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 is an RNA-guided genome editing tool that enables targeted DNA cleavage using a single guide RNA (sgRNA) and the Cas9 endonuclease1 . A key advancement involves using two sgRNAs to excise DNA segments between cut sites, generating more reliable knockouts2 . This project aims to develop and optimize the CRISPR/Cas9 knockout (KO) lentivector system containing two sgRNA cassettes. Typical knockout vectors using one sgRNA produce shorter (<20 bp) insertions/deletions (indels). We hypothesize that a dual-sgRNA vector will generate larger indels at a specific genomic locus, more effectively disrupting protein production. The Civin lab3 showed that SIX1 overexpression in TF1 and CD34+ hematopoietic stem-progenitor cells (HSPCs) enhances erythroid differentiation and GATA1 expression, while suppressing GATA2 and CD34. In contrast, CRISPR/Cas9-mediated SIX1 knockout impairs erythropoietin-stimulated erythropoiesis, revealing cooperation between the PAX-SIX-EYA-DACH and GATA networks. We have constructed lentiviral vectors to selectively CRISPR/Cas9 knockout SIX1 in TF1 and HEK293T cells. sgRNAs that selectively disrupt SIX1 genomic targets have been validated by Inference of CRISPR Edits (ICE) analysis of genomic DNA amplicon sequences and Western immunoblots. Puromycin selection and green fluorescent protein fluorescence activated cell sorting (GFP FACS) will be used to select and identify transduced cells. These studies aim to clarify how SIX isoforms and its interacting molecules (e.g., GATA1) influence human hematopoiesis, with implications for ex vivo erythropoiesis in transfusion and transplantation.