Abstract: Some aspects provide for techniques for genotyping cells in a biological sample. In some embodiments, the techniques include: obtaining single cell DNA sequence (scDNA-seq) data for a plurality of droplets; and genotyping the cells using the scDNA-seq data, the genotyping comprising determining for each droplet, a genotype for a locus of a respective genome of at least one cell associated with the droplet, the determining comprising: identifying, from among the plurality of droplets, a first set of droplets associated with cells that are homozygous at the locus; identifying, from among the plurality of droplets not in the first set of droplets, a second set of droplets associated with more than two alleles at the locus; and identifying, from among the plurality of droplets not in the first or second sets of droplets, a third set of droplets associated with cells that are heterozygous at the locus.

Abstract: Provided herein are methods of making genetically engineered cells having a plurality of modifications (e.g., insertions or deletions), cells and cell populations produced by said methods, methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Provided herein are chimeric antigen receptors (CARs) with binding specificity for CD123. Nucleic acids, expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the CARs are also disclosed, and methods including the treatment of a hematopoietic malignancy or premalignancy characterized by the expression of CD123, e.g., leukemias such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

Abstract: The disclosure features methods of reducing or inhibiting an anti-drug antibody response in a subject, as well as methods of reducing or inhibiting unwanted immune cell activation in a subject to be treated with a messenger RNA (mRNA), comprising administering to the subject a mRNA, e.g., a chemically modified messenger RNA (mmRNA), encoding a polypeptide of interest, wherein the mRNA comprises at least one microRNA (miR) binding site for a miR expressed in immune cells, such as miR-126 binding site and/or miR-142 binding site, such that an anti-drug antibody response to the polypeptide or interest, or unwanted immune cell activation (e.g., B cell activation, cytokine secretion), is reduced or inhibited in the subject. The disclosure further provides therapeutic treatment regimens designed to reduce or inhibit ADA or unwanted immune cell activation (e.g., B cell activation, cytokine secretion) in a subject being treated with mRNA-based therapeutics.

Abstract: Provided herein, in some embodiments, are methods and compositions for treatment of subjects with ?-thalassemia and subjects with severe sickle cell disease using autologous CRISPR-Cas9 modified CD34+ human hematopoietic stem and progenitor cells.

Abstract: Provided herein are fusion polypeptides comprising a Cpf1 domain lacking nuclease activity and an endonuclease domain. Also provided herein are fusion polypeptides further comprising a genomic modification domain, which in some embodiments is a base editor, such as a deaminase. Also provided herein are methods involving contacting the fusion polypeptides with a gRNA to form a genetic editing system directed to a target site sequence in the genome of a cell.

Abstract: When a cancer patient is administered an anti-cancer therapy targeting a lineage specific cell-surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and/or CD312 (EMR2)), e.g., in the form of an immunotherapeutic agent, the therapy can deplete not only cancer cells expressing the lineage-specific cell-surface antigen, but also noncancerous cells expressing the lineage-specific cell-surface antigen in an “on-target, off tumor” effect. This disclosure provides, e.g., novel cells having a modification (e.g., insertion or deletion) in an endogenous lineage-specific cell-surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and/or CD312 (EMR2)) gene. The disclosure also provides compositions, e.g., gRNAs, that can be used to make such a modification.

Abstract: Provided herein, in some embodiments, are methods and compositions for treatment of subjects with ?-thalassemia and subjects with severe sickle cell disease using autologous CRISPR-Cas9 modified CD34+ human hematopoietic stem and progenitor cells.

Abstract: Provided herein are oligonucleotides comprising a first region that is complementary to a targeting domain of a gRNA and a second region that is complementary to a CRISPR RNA (crRNA) sequence for a CRISPR/Cas nuclease, wherein the oligonucleotide reduces genomic editing at a target sequence complementary to the targeting domain of the gRNA. Also provided herein are methods involving contacting a gRNA, a CRISPR/Cas nuclease, a complex comprising the same, or a cell comprising any thereof with such oligonucleotides.

Abstract: Provided herein are IL-2 reporter systems comprising nucleic acid constructs comprising a nucleotide sequence encoding a reporter molecule operably linked to a minimal nuclear factor of activated T cells (NFAT)-responsive promoter. Also provided herein are vectors, cells, and cell lines comprising such nucleic acids. Also provided herein are methods of making and using such cells, for example to measure the ability of a chimeric antigen receptor (CAR) to induce nuclear factor of activated T cells (NFAT)-signaling in a cell.

Abstract: Provided herein are gRNA comprising a targeting domain that targets BCMA, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the BCMA gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Materials and methods for treating a patient with a hemoglobinopathy, both ex vivo and in vivo, and materials and methods for deleting modulating or inactivating a transcriptional control sequence of a BCL11A gene in a cell by genome editing.

Abstract: The invention relates to compositions and methods for the manufacture and optimization of modified mRNA molecules via optimization of their terminal architecture.

Abstract: Provided herein are gRNA comprising a targeting domain that targets CD5, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the CD5 gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Provided herein are gRNA comprising a targeting domain that targets CD7, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the CD7 gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Aspects of the disclosure provide methods and compositions for treating a hematopoietic malignancy (e.g., acute myeloid leukemia). In some aspects, the disclosure provides methods of treatment using a population of genetically engineered CD33-deficient hematopoietic cells and a cytotoxic agent comprising an anti-CD33 antigen-binding domain.

Abstract: The disclosure is directed to methods and compositions relating to genetically engineered cells expressing chimeric antigen receptors, where the cells are mobilized lymphocytes.

Abstract: Provided herein are gRNA comprising a targeting domain that targets CD38, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the CD38 gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Provided herein are gRNA comprising a targeting domain that targets CD6, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the CD6 gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.

Abstract: Provided herein are gRNA comprising a targeting domain that targets CD30, which may be used, for example, to make modifications in cells. Also provided herein are methods of genetically engineered cell having a modification (e.g., insertion or deletion) in the CD30 gene and methods involving administering such genetically engineered cells to a subject, such as a subject having a hematopoietic malignancy.