Base editing and crispr/cas9 gene editing strategies to correct cd3 severe combined immunodeficiency in hematopoietic stem cells
AssigneeThe Regents of the University of California
InventorsDonald B. KOHN, Grace MCAULEY
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Abstract
Provided herein are compositions, systems, and methods to provide two gene editing-based approaches that can be used to correct the CD35 SCID-causing C202T mutation (TGA→CGA). In certain embodiments one approach involves CRISPR/Cas9 homology-directed repair (HDR)-mediated correction with a single-strand oligodeoxynucleotide (ssODN) homologous donor. In certain embodiments another approach comprises Adenine Base Editing (ABE)-correction, to precisely revert the CD35 SCID-causing C202T mutation (TGA→CGA).
Claims67
A system for homology-directed repair (HDR)-mediated correction of the C202T mutation that produces CD3δ SCID disease, said system comprising:
- a first single-guide RNA (sgRNA) that directs Cas9 cutting upstream of the C2020T mutation;
- a second single-guide RNA (sgRNA) that directs Cas9 cutting downstream of the C2020T mutation;
- and
- a single-strand oligodeoxynucleotide (ssODN) homologous donor comprising a nucleotide sequence that corrects the C202T mutation.
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An adenosine base editor, wherein said base editor is a variant of the wildtype NGG-recognizing Cas9 (D10A) nickase (Cas9n) comprising a combination of amino acid substitutions selected from the group consisting of:
- (1) NRTH-ABE8e:
- A10T, I322V, S409I, E427G, R654L, R753G, R1114G, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, E1253K, P1321S, D1332G, and R1335L;
- (2) VRER-ABE8e:
- D1135V, G1218R, R1335E, and T1337R;
- and
- (3) A262T, R324L, S409I, E480K, E543D, M694I, and E1219V.
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A lentivirus for evaluating gene editing correction of the CD3δ SCID-causing C202T mutation, said lentivirus construct comprising the elements illustrated in FIG. 3.
Dependencies are inferred from the claim text.