The present disclosure generally relates to evolved cytidine deaminases derived from cytidine deaminases, and methods of editing DNA using the same. In some aspects, the disclosure describes the directed evolution of a TadA-derived adenosine deaminase (TadA-CD) to perform cytidine deamination. In some embodiments, the TadA-CDs comprise a plurality of mutations compared to the parent TadA variant. In some embodiments, the TadA-CD is fused to a programmable DNA binding protein. Other aspects of the disclosure generally relate to a cytosine base editor (CBE) comprising a programmable DNA binding protein and the TadA-CD. In some embodiments, the disclosed cytosine base editor has improved efficiencies of conversion and reduced off-target editing frequencies compared to naturally-occurring CBEs. Also provided are polynucleotides, vectors, and kits useful for the generation and delivery of the CBEs. Cells containing such vectors and CBEs are also provided. Further provided are methods of treatment comprising administering the CBEs.

Embodiments of the disclosure encompass improvements on cell therapies by allowing the cells to be more effective for cancer treatment, including in a solid tumor microenvironment. In specific cases, the cells are modified to have reduced or inhibited levels of expression of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, or CREB1, such as by CRISPR gene editing. In certain cases, the cells are modified to have reduced or inhibited levels of expression of CREM. In certain cases, the cells are further modified to express, for example, one or more engineered receptors, one or more cytokines, and/or optionally one or more suicide genes.

The technology described herein relates to circularized prime editing guide RNAs (cpegRNAs) comprising at least a spacer, a gRNA scaffold, a primer binding site, and a template sequence with one or more nucleotide changes relative to a target sequence. The disclosure also provides compositions and prime editing systems comprising the pegRNAs and uses thereof for prime editing.

The present disclosure provides methods and compositions for genetically modifying hematopoietic stem and progenitor cells (HSPCs), in particular by replacing the HBA1 or HBA2 locus in the HSPCs with a transgene encoding a therapeutic protein.

The present disclosure provides Type VI CRISPR-Cas effector polypeptides that can, when complexed with a guide nucleic acid, modify a target RNA. A Type VI CRISPR-Cas effector polypeptide of the present disclosure can also provide for detection of nucleic acid by cleavage of non-target RNAs. The present disclosure provides methods of modifying a target RNA, and methods of detecting a nucleic acid.

Disclosed herein are immune effector cells for use in adoptive cell transfer that have chemically- or genetically-inhibited PDHB (Pyruvate dehydrogenase E1 subunit beta) expression or activity. Also disclosed are methods of inhibiting or ablating PDHB expression in immune effector cells ex vivo and methods of using these cells to treat subjects with cancer. In some embodiments, the immune effector cells are further treated with a TIMS inhibitor or genetically engineered to ablate TIM3 expression. In some embodiments, the immune effector cells are further treated with a LAGS inhibitor or genetically engineered to ablate LAG3 expression. In some cases, the PDHB gene is disrupted by insertion of the gene encoding the chimeric receptor into the PDHB gene loci of the cell. Therefore, disclosed herein is a chimeric cell expressing a chimeric receptor.

Provided herein are compositions for gene modification related to base editor systems, and methods of using the same to treat or prevent conditions associated with the extracellular deposition in various tissues of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) proteins. Such conditions include, but are not limited to, polyneuropathy due to hereditary transthyretin amyloidosis (hATTR-PN) and hereditary cardiomyopathy due to transthyretin amyloidosis (hATTR-CM), both associated with autosomal dominant mutations of the TTR gene, and an age-related cardiomyopathy associated with wild-type TTR proteins (ATTRwt), also known as senile cardiac amyloidosis.

The present disclosure provides compositions and methods for modifying a PCSK9 gene. In some aspects, the present disclosure provides a guide RNA, compositions thereof, and pharmaceutical compositions comprising a guide RNA or a composition as described herein. In some aspects, the present disclosure also provides uses and methods of using a guide RNA, a composition thereof, or a pharmaceutical composition as described herein, for inducing a double-strand break or a single-strand break in a PCSK9 gene, for reducing expression of a PCSK9 gene in a cell or subject, and for treating a patient having or at risk of having a PCSK9-related disease or condition.

Compositions and methods for effecting base editing to correct mutations in the phenylalanine hydroxylase gene, thereby curing phenylketonuria, are disclosed.

There are provided in vitro and in vivo methods of editing the C9ORF72 repeat expansion mutation using a nuclease to edit a nucleic acid in which the expansion is found. An exemplary method uses a Cas-9 editing system. Guide nucleic acids for editing the repeat expansion mutation are provided. Also provided is a method of mitigating or eliminating symptoms arising in a subject due to the presence of the mutation in the subject's genome.