The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.

A method for producing genetically engineered immune cells, e.g. T cells, or iPSCs which uses an RNA-scaffold mediated base editing system. The method enables precise modifications to be made to the genome whilst minimizing the possibility of off-target effects, making the method particularly suitable for therapeutic applications.

The present invention relates to the analysis of complex single cell sequencing libraries. Disclosed are methods for enrichment of library members based on the presence of cell-of origin barcodes to identify and concentrate DNA that is relevant to interesting cells or components that would be expensive or difficult to study otherwise. Also, disclosed are methods of capturing cDNA library molecules by use of CRISPR systems, hybridization or PCR. The present invention allows for identifying the properties of rare cells in single cell RNA-seq data and accurately profile them through clustering approaches. Further information on transcript abundances from subpopulations of single cells can be analyzed at a lower sequencing effort. The methods also allow for linking TCR alpha and beta chains at the single cell level.

The present invention relates to a utilizing method of a nucleic acid compound containing a selectively cleavable site. Also, the present invention relates to a DNA-encoded library containing the selectively cleavable site, a composition for synthesis therefor and a method of use thereof.

Some aspects of this disclosure provide compositions, strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.

Provided is a mutant UDG having improved thermal sensitivity compared to a wild-type UDG. The mutant UDG of the presently claimed subject matter having a high thermal sensitivity has no inhibitory effect on the PCR reaction and thus can be advantageously used for the development of PCR/qPCR Premix and particularly PCR diagnostic kits employing UDG which requires the use of relatively low temperature in melting and amplification steps.

The present invention discloses base editing systems for mutating a base C to A and a base C to G and applications thereof. The base editing system for mutating C to A disclosed in the present invention includes cytosine deaminase AID and nCas9 nuclease or includes cytosine deaminase AID, nCas9 nuclease and uracil DNA glycosidase; the base editing system for mutating C to G of the present invention includes cytosine deaminase APOBEC, nCas9 nuclease and uracil DNA glycosidase. The experiments show that a combination of the three base editing systems for mutating C to A, C to T and A to G can realize a mutation of A, T, C or G to any base in both prokaryotes and eukaryotes.

The type V-U1 system from the bacterium Mycobacterium mucogenicum CCH10-A2 (Mmu) has a nuclease which binds dsDNA but it does not cleave it. Additionally, after dsDNA binding by the nuclease an RuvC-dependent interference of nascent transcript (mRNA) takes place and this mechanism has not been described before for any CRISPR system. CRISPR based gene manipulation can therefore use CRISPR endonucleases from the Type V-U1 system from Mycobacterium mucogenicum, including variant and modified endonucleases, so as to provide for methods of expression control and gene editing in cells of any living organism or of any nucleic acid in vitro.

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

The invention relates to improved methods of enzymatic solid-supported nucleic acid synthesis that make use of terminal deoxynucleotidyl transferase (TdT) enzymes or modified terminal deoxynucleotidyl transferase (TdT) enzymes on polyacrylamide type supports. The invention further relates to the use of kits comprising said enzymes in a method of solid-supported nucleic acid synthesis.