Altering gene expression in cart cells and uses thereof

A method of generating a CRISPR-modified T cell, the method comprising introducing into a T cell:

• a CRISPR system that generates an insertion or deletion in an endogenous TCR α chain (TRAC gene locus, an endogenous TCR β chain (TRBC) gene locus, causing downregulated gene expression of the endogenous TRAC gene and the endogenous TRBC gene,
• (b) a CRISPR system that generates an insertion or deletion in an endogenous beta 2-microglobulin (B2M) gene locus causing downregulated gene expression of the endogenous B2M gene
• wherein the CRISPR system comprises a CRISPR-associated (Cas) nuclease and a guide RNA, and wherein:
• (i) first guide RNA comprises a nucleic acid sequence capable of targeting nucleic acid sequence of SEQ ID NO:
• 15 within the endogenous TRAC gene;
• and
• (ii) second guide RNA comprises a nucleic acid sequence capable of targeting a nucleic acid sequence of SEQ ID NO:
• 16 within the endogenous TRBC gene
• (iii) a third guide RNA comprises a nucleic acid sequence capable of targeting a nucleic acid sequence of SEQ ID NO:
• 42 within the endogenous B2M gene, thereby respectively downregulating gene expression of the endogenous TRAC gene, TRBC gene, and B2M gene in the T cell;
• and
• a nucleic acid encoding a chimeric antigen receptor (CAR), thereby generating the CRISPR-modified T cell.