The present invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for creating novel herbicide resistant plants by base editing techniques and a method for screening endogenous gene mutation sites capable of conferring herbicide resistance in plants. The invention also relates to the use of the identified endogenous gene mutantation sites in crop breeding.

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

The present disclosure provides methods of introducing site-specific mutations in a target cell and methods of determining efficacy of enzymes capable of introducing site-specific mutations. The present disclosure also provides methods of providing a bi-allelic sequence integration, methods of integrating of a sequence of interest into a locus in a genome of a cell, and methods of introducing a stable episomal vector in a cell. The present disclosure further provides methods of generating a human cell that is resistant to diphtheria toxin.

Provided is a method for editing a target RNA at a target residue position of a host cell. The method comprises introducing ADAR-recruiting RNA (arRNA) or a construct encoding the arRNA into a host cell, wherein the arRNA comprises a complementary RNA sequence that hybridizes to a target RNA; the target residue is located in a three-base motif comprising a 5′ nearest-neighbor residue (upstream residue) of the target residue in the target RNA, the target residue, and a 3′ nearest-neighbor residue (downstream residue) of the target residue in the target RNA, wherein the three-base motif is not UAG, and the complementary RNA sequence comprises a mismatch directly opposite to the upstream residue or the downstream residue in the target RNA. Further provided are arRNA for the method, an RNA obtained by the method, a host cell comprising the RNA, and use of the method in the treatment of a disease.

Provided are an improved genome editing system and a method that has high specificity, which are capable of obtaining stable mutation types. The system includes an expression construct including a coding sequence of a gRNA targeting at least one genomic target sequence; an expression construct including a coding sequence of a CRISPR nuclease; and an expression construct including a coding sequence for a gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease. Upon introduction into the cell, the gRNA targeting the at least one genomic target sequence directs the CRISPR nuclease to the at least one genomic target sequence and results in one or more mutations in the genomic target sequence, and the gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease directs the CRISPR nuclease to the target sequence within the coding sequence of the CRISPR nuclease and results in an inactivating mutation of the CRISPR nuclease.

The present invention relates to a base editing compound comprising or consisting of (a) a Cas protein, and, covalently connected therewith; (b) a nucleobase-modifying enzyme, wherein the covalent connection of (a) and (b) is (i) direct; (ii) provided by a peptide comprising at least one Pro residue, said peptide having a length between 1 and 20 preferably between 1 and 15 amino acids; or (iii) provided by a non-peptidic linker, said non-peptidic linker being a small organic molecule comprising one or more double bonds, one or more triple bonds, and/or one or more aromatic rings.

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 19, 24, 26, 27, 28, 33, 40, 49, 50, 63, 95, 96, 97, and/or 99 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.

As described below, the present invention features genetically modified immune cells having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction, or a combination thereof. The present invention also features methods for producing and using these modified immune effector cells.

The invention features nucleobase editors and multi-effector nucleobase editors having an improved editing profile with minimal off-target deamination, compositions comprising such editors, and methods of using the same to generate modifications in target nucleobase sequences.