The disclosure provides a novel design of guide RNA and uses thereof. In one aspect, the guide RNA comprises a spacer sequence flanked by a direct repeat sequence at both the 5′ end and the 3′ end of the spacer sequence.

The present invention features genetically modified immune cells comprising novel adenosine base editors (e.g., ABE8) having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft-versus-host reaction or host-versus-graft reaction, or a combination thereof. The present invention also features methods for producing and using these modified immune effector cells.

The present invention features compositions and methods for editing deleterious mutations associated with hemoglobinopathies, such as sickle cell disease (SCD). In particular embodiments, the invention provides methods for correcting mutations in a beta globin polynucleotide using modified adenosine base editors termed “ABE8” having unprecedented levels (e.g., >60-70%) of efficiency.

The disclosure provides adenosine deaminases that are capable of deaminating adenosine in DNA to treat cancers, such as melanoma and glioblastoma. The disclosure also provides fusion proteins, guide RNAs and compositions comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA, for example in a STAT3 gene. In some embodiments, adenosine deaminases provided herein are used to modify the STAT3 gene so that its protein product, STAT3, is unable to be activated. In some embodiments, the methods and compositions provided herein are used to treat melanoma or glioblastoma.

Aspects of the disclosure relate to compositions and methods for exon replacement in a cell or a subject. In some embodiments, the disclosure relates to isolated nucleic acids (and vectors, such as rAAV vectors) encoding one or more guideRNAs (gRNAs) that target an intron-exon boundary; an intronic sequence having a splice signal; and a donor sequence encoding a gene product of a gene of interest, or portion thereof. In some embodiments, compositions described herein are useful for replacing mutant exons associated with certain diseases, for example Duchen's muscular dystrophy (DMD), cystic fibrosis (CF), spinal muscular atrophy (SMA), Rett syndrome, and mucopolysaccharidosis (MPS).

Provided is a method for deaminating targeted cytosine in a target RNA of a cell. The method comprises: introducing, into the cell, a modified adenosine deaminase protein or a catalytic domain thereof, or a construct expressing the modified adenosine deaminase protein or the catalytic domain thereof, and a construct comprising an arRNA oligonucleotide that recruits the modified adenosine deaminase protein or the catalytic domain thereof to the target RNA or expressing arRNA. Further provided are an engineered composition or system for RNA editing, and a use of treating a disease by using the engineered composition or system to correct a T-C mutation.

The present application relates to an LEAPER-based method for targeted editing of RNA, which comprises: safely and effectively performing adenosine-to-inosine RNA editing (A-to-I RNA editing) by LEAPER in vivo, thereby the pathogenic mutation sites can be accurately repaired, and all diseases caused by G>A mutations, such as MPS IH, can be treated.

RNA editing tools for use in systems designed to measure RNA in vivo and manipulate specific cell types are disclosed herein. An RNA sensor system comprising a) a single-stranded RNA (ssRNA) sensor comprising a stop codon and a payload; optionally wherein the ssRNA sensor further comprises a normalizing gene; and b) an adenosine deaminase acting on RNA (ADAR) deaminase; wherein the sensor is capable of binding to a ssRNA target to form a double-stranded RNA (dsRNA) duplex that becomes a substrate for the ADAR deaminase; wherein the substrate comprises a mispairing within the stop codon; and wherein the mispairing is editable by the ADAR deaminase, which editing can effectively remove the stop codon so as to enable translation and expression of the payload. A method of quantifying ribonucleic acid (RNA) levels using the RNA sensor system is also disclosed.

An oligonucleotide that can induce editing activity specific to adenosine deaminase-1. The oligonucleotide includes a first oligonucleotide for identifying a target RNA and a second oligonucleotide linked to the 5′ side of the first oligonucleotide, and that induces site-specific editing of the target RNA. The first oligonucleotide includes a target-corresponding nucleotide residue that corresponds to an adenosine residue in the target RNA; an oligonucleotide comprising 10-24 residues that is linked to the 3′ side of the target-corresponding nucleotide residue and has a base sequence which is complementary to the target RNA; and an oligonucleotide comprising 3-6 residues that is linked to the 5′ side of the target-corresponding nucleotide residue and has a base sequence which is complementary to the target RNA. The second oligonucleotide comprises 2-10 residues and forms a complementary strand to the target RNA, and the complementary strand includes at least one mismatched base

Provided are methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA, deaminase-recruiting RNAs used in the RNA editing methods, compositions and kits comprising the same.