Nucleic acids and viral vectors, particularly adeno-associated virus (AAV) vectors are provided that encode Cas9 and paired guide RNAs. The nucleic acids and vectors, and compositions that comprise them, can be used in methods to treat subjects, to alter cells in subjects who may suffer from an inherited retinal dystrophy such as CEP290 associated disease or who may be in need of alteration of a cell or a cellular nucleic acid sequence associated with an inherited retinal dystrophy such as the CEP290 gene, and/or to treat inherited retinal dystrophies including CEP290 associated disease.

The disclosure relates to methods and compositions for regulating expression of DUX4. Specifically, the disclosure provides a recombinant gene editing complex comprising: a recombinant gene editing protein; and, a nucleic acid encoding a guide RNA (gRNA) that specifically hybridizes to a target nucleic acid sequence encoding a D4Z4 macrosatellite repeat region, wherein binding of the complex to the target nucleic acid sequence results in inhibition of DUX4 gene expression. In some aspects, methods described by the disclosure are useful for treating a disease associated with aberrant DUX4 expression (e.g., facioscapulohumeral muscular dystrophy, FSHD).

Materials and methods for treating a patient with a hemoglobinopathy, both ex vivo and in vivo, and materials and methods for creating permanent changes to the genome that can result in at least one deletion, insertion, modulation, or inactivation of a transcriptional control sequence of a BCL11A gene in a cell by genome editing.

Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g., mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivo to the host (e.g., mammalian) cell.

An Argonaute protein from eukaryotes and an application thereof are provided. An amino acid sequence of the Argonaute protein is shown in SEQ ID NO: 1 or has at least 50% sequence identity with the sequence shown in SEQ ID NO: 1. The specific cleavage activity of the eukaryotic Argonaute protein on DNA is first proved, and an experimental proof for the study of interaction between the eukaryotic Argonaute protein and DNA is provided. In addition, polypeptides, nucleic acids, expression vectors, compositions, kits, and methods used therein can carry out site-specific operation on intracellular and extracellular genetic materials and can be effectively applied in many fields of biotechnology, providing a new tool for gene editing, modification, and molecular detection of Argonaute polypeptides based on eukaryotic sources.

Compositions and use of the compositions in methods of detecting SARS-CoV-2 in a sample is disclosed, using RT-LAMP coupled with CRISPR-Cas12, referred to herein as iSCAN (in vitro Scanning of COVID-19-Associated Nucleic acids) is disclosed. iSCAN provides a rapid, specific, accurate, sensitive detection of SARS-CoV-2 in a sample. The iSCAN is 1) rapid, as the RT-LAMP and CRISPR-Cas12/Cas 13 reaction takes less than 1 h; 2) specific, because detection depends on the identification and subsequent cleavage of SARS-CoV-2 genomic sequences by the Cas12 or 13 enzyme; 3) field-deployable, as only simple equipment is required; and 4) easy to use, as the colorimetric reaction coupled to lateral flow immunochromatography makes the assay results easy to assess. The methods include amplifying SARS-CoV-2 in a sample using RT-LAMP and using the RT-LAMP product as a substrate in a CRISPR-Cas12/13 reaction, incorporated with a means of detecting the presence of the SARS-CoV-2 RT-LAMP product.

A nucleic acid construct is disclosed which is removable after transformation. Methods of using same are disclosed as well.

The disclosure features methods of correcting a mutation in the human beta-globin (HBB) gene in a cell or population of cells. The disclosure also features methods of increasing repair of a DNA double stranded break (DSB) in an HBB gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.

The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides Cas proteins and their use in modifying target sequences.

Provided herein are compositions and methods for improving the genome-wide specificities of targeted base editing technologies.