Nuclease-mediated genome editing of primary cells and related kits

A method for stably integrating via homologous recombination a polynucleotide sequence into the HBB gene of one or more primary hematopoietic stem or progenitor cells (HSPCs) of a population of HSPCs isolated from a subject having sickle cell disease, the method comprising introducing into the one or more primary HSPCs:

• (a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid sequence in the HBB gene of the one or more primary cells and a second nucleotide sequence that interacts with a CRISPR-associated Cas polypeptide, wherein the first nucleotide sequence, the second nucleotide sequence, or both comprise a 2′-O-methyl 3′-phosphorothioate nucleotide;
• (b) the Cas polypeptide, wherein the modified sgRNA and the Cas polypeptide are formed in a ribonucleoprotein (RNP) complex prior to introducing into the one or more primary cells, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid sequence in the HBB gene;
• and
• (c) a recombinant adeno associated viral vector serotype 6 (AAV6) comprising the polynucleotide sequence,
• wherein the Cas polypeptide cleaves the target nucleic acid sequence in the HBB gene, thereby generating a double-strand break in the target nucleic acid sequence;
• and
• wherein the polynucleotide sequence is stably integrated into the target nucleic acid sequence in the HBB gene of greater than 10% of the primary HSPCs in the population of primary HSPCs.

A kit comprising a population of primary cells, wherein one or more primary cells of the population comprises:

• (a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid sequence in the genome of the one or more primary cells and a second nucleotide sequence that interacts with a CRISPR-associated Cas polypeptide, wherein the first nucleotide sequence, the second nucleotide sequence, or both comprise a 2′-O-methyl 3′-phosphorothioate nucleotide;
• (b) a Cas polypeptide, wherein the modified sgRNA and the Cas polypeptide are formed in a ribonucleoprotein (RNP) complex, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid sequence;
• and
• (c) a recombinant adeno associated viral vector, serotype 6 (rAAV6) comprising a recombinant donor template comprising two nucleotide sequences comprising two non-overlapping, homologous portions of the target nucleic acid, wherein the nucleotide sequences are located at the 5′ and 3′ ends of a nucleotide sequence corresponding to the target nucleic acid to undergo homologous recombination;
• wherein a nucleotide sequence of the donor template is stably integrated into the target nucleic acid sequence in the genome of greater than 10% of primary cells in the population of primary cells.