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.

A high-fidelity AsCpf1 mutant is obtained by performing a mutation on arginine at position 951 and/or 955 of a AsCpf1 protein amino acid sequence and replacing the same with an amino acid free of forming a hydrogen bond with DNA of a target site; and the amino acid sequence thereof is shown in SEQ ID NOS: 1-3. The encoding gene of the AsCpf1 mutant has a nucleotide sequence as shown in SEQ ID NO: 4 and can be used in the construction of a CRISPR/AsCpf1 gene editing system. A CRISPR/AsCpf1 gene editing system includes a gene encoding a AsCpf1 protein, and the AsCpf1 protein is the AsCpf1 mutant mentioned above. The CRISPR/AsCpf1 gene editing system can be used in lowering an off-target effect of gene editing. The novel AsCpf1 mutant not only retains the gene editing efficiency of wild-type AsCpf1, but also has a higher specificity than the wild-type AsCpf1.

Genetically encoded calcium indicator (GECI) polypeptides and the nucleic acid molecules encoding such polypeptides are provided.

The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.

The present invention relates to the production and use of covalently closed circular (ccc) recombinant plasmids, and more particularly to vector modifications that improve expression of said DNA molecules in the target organism.

Provided are methods and compositions for generating a deletion library, and methods and compositions for generating and identifying a defective interfering particle (DIP). Also provided are transposon cassettes. A subject method can include: inserting a transposon cassette comprising a target sequence for a sequence specific DNA endonuclease into a population of circular target DNAs to generate a population of transposon-inserted circular target DNAs; contacting the population of transposon-inserted circular target DNAs with the sequence specific DNA endonuclease to generate a population of cleaved linear target DNAs; contacting the population of cleaved linear target DNAs with one or more exonucleases to generate a population of deletion DNAs; and circularizing the deletion DNAs to generate a library of circularized deletion DNAs. The population of circular target DNAs can include viral genomic DNA. Also provided are human immunodeficiency virus (HIV) deletion mutants, e.g., interfering, conditionally replicating, HIV deletion mutants, and related constructs.

Methods and constructs for engineering circular RNA are disclosed. In some embodiments, the methods and constructs comprise a vector for making circular RNA, the vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, the vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. Methods for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector are also disclosed.

Described are engineered cells that include genetic alterations leading to up- or down-regulation of certain genes in the cells for improved production of a recombinant protein, especially one that is not easily expressed at high levels in unaltered cell lines. Also provided are methods of preparing and using such cells.

This invention pertains to a novel population of lymphocytes, methods for producing these, and their use in the treatment of diseases.

According to one embodiment, a vector set includes a first vector and a second vector. The first vector includes a transposase target sequence, a first promoter sequence ligated to downstream of the transposase target sequence, and a first reporter gene ligated to downstream of the first promoter sequence. The second vector includes a 5′-side transposase recognition sequence, a 3′-side transposase recognition sequence, and a first enhancer sequence arranged therebetween.