Dna-containing polynucleotides and guides for crispr type v systems, and methods of making and using the same

A method for producing a chimeric antigen receptor (CAR)-expressing cell, said method comprising:

• a) contacting a first target nucleic acid comprising a TRAC sequence in a cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a first CRISPR guide molecule having a targeting region capable of binding the first target nucleic acid sequence;
• and an activating region capable of forming a nucleoprotein complex with the Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base in the activating region, the targeting region, or both, and the nucleoprotein complex is capable of cleaving the first target nucleic acid sequence;
• b) contacting a second target nucleic acid sequence comprising a B2M sequence in the same cell with a nucleoprotein complex comprising a catalytically active Cas12 protein and a second CRISPR guide molecule having a targeting region capable of binding the second target nucleic acid sequence;
• and an activating region capable of forming a nucleoprotein complex with the Cas12 protein, wherein said CRISPR guide molecule comprises ribonucleotide bases and at least one deoxyribonucleotide base in the activating region, the targeting region, or both, and the nucleoprotein complex is capable of cleaving the second target nucleic acid sequence;
• c) providing a first donor polynucleotide encoding a CAR comprising an scFv, a transmembrane domain, a co-stimulatory domain, and an activating domain, wherein the CAR is capable of being inserted into the cleavage site in the first target nucleic acid sequence;
• d) providing a second donor polynucleotide encoding a B2M-HLA-E fusion construct comprising a B2M secretion signal, a HLA-G peptide signal sequence, a first linker sequence, a B2M sequence, a second linker sequence, and a HLA-E sequence, wherein the B2M-HLA-E fusion construct is capable of being inserted into the cleavage site in the second target nucleic acid sequence;
• e) cleaving the first target nucleic acid sequence and inserting at least a portion of the first donor polynucleotide into the cleavage site;
• and
• f) cleaving the second target nucleic acid sequence and inserting at least a portion of the second donor polynucleotide into the cleavage site.