Universal donor cells

An in vitro method for generating a universal donor cell, the method comprising delivering to a stem cell:

• (a) a ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) gene locus, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:
• 2;
• and
• (b) a vector comprising a nucleic acid, the nucleic acid comprising (i) a nucleotide sequence homologous with a region located left of the target site in the B2M gene locus, wherein the nucleotide sequence consists essentially of SEQ ID NO:
• 13, (ii) a nucleotide sequence encoding a tolerogenic factor, and (iii) a nucleotide sequence homologous with a region located right of the target site in the B2M gene locus, wherein the nucleotide sequence consists essentially of SEQ ID NO:19, wherein the vector comprises a nucleotide sequence consisting of SEQ ID NO:
• 33,
• wherein the B2M gene locus is cleaved at the target site and the nucleic acid is inserted into the B2M gene locus within 50 base pairs of the target site, thereby disrupting the B2M gene and generating the universal donor cell, wherein the universal donor cell has increased immune evasion and/or cell survival compared to a stem cell that does not comprise the nucleic acid inserted into the B2M gene locus.