Compositions and methods for T-cell receptor gene assembly

A method for generating a plurality of at least 100 nucleic acid molecules encoding a plurality of at least 100 different native T-cell receptor (TCR) alpha chains or portions thereof from a mixed population of cells in a single compartment, the method comprising:

• (a) providing a first plurality of at least 100 nucleic acid molecules in a single compartment, each nucleic acid molecule of the first plurality of at least 100 nucleic acid molecules comprising a sequence encoding a CDR3 alpha (CDR3a) of a different TCR alpha chain and a unique connector sequence;
• (b) providing a second plurality of nucleic acid molecules in the same single compartment containing the first plurality of at least 100 nucleic acid molecules of (a), thereby contacting the first plurality of at least 100 nucleic acid molecules of (a) to the second plurality of nucleic acid molecules in the same single compartment, wherein each nucleic acid molecule of the second plurality of nucleic acid molecules comprises a sequence from a TCR alpha variable (TRAV) gene and a unique anti-connector sequence, and wherein the second plurality of nucleic acid molecules comprises at least five different sequences from at least five different TRAV genes;
• and
• (c) generating within the same single compartment a third plurality of at least 100 nucleic acid molecules, each nucleic acid molecule of the third plurality comprising a sequence from one of the sequences encoding a CDR3a from the first plurality of at least 100 nucleic acid molecules of (a) and a sequence from one of the at least five different TRAV genes of the second plurality of nucleic acid molecules of (b), wherein generating comprises specifically linking the unique connector sequence of a nucleic acid molecule of the first plurality of at least 100 nucleic acid molecules of (a) to the unique anti-connector sequence of a nucleic acid molecule of the second plurality of nucleic acid molecules of (b) that comprises a sequence from a single given TRAV gene of the at least five different TRAV genes, thereby generating the plurality of at least 100 nucleic acid molecules encoding the plurality of at least 100 different native TCR alpha chains or portions thereof from the mixed population of cells, wherein the unique anti-connector sequence of the sequence from the single given TRAV gene is complementary to the unique connector sequence of the nucleic acid molecule of the first plurality of at least 100 nucleic acid molecules, and wherein the single given TRAV gene is cognate to the CDR3a to which it is linked.