The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3 or a 5 primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3 flanking sequence and a universal 5 flanking sequence, respectively. The methods involve performing a first amplification reaction on the plurality of pairs of oligonucleotides; removing the 3 and 5 primer binding sequences from the plurality of pairs of oligonucleotides; and subjecting the plurality of pairs of oligonucleotides to an assembly reaction to thereby assemble the dsDNA molecule having the desired sequence.

This invention provides methods for detecting RNAs in a biological sample while using only a small amount of sample input, e.g., less than or equal to 1 mL. The methods described herein are able to detect a large percentage of protein-coding genes having the ENSEMBL gene annotation HG38.

The present invention provides a complex containing a nucleic acid sequence-recognizing module and a proteolysis tag, wherein the module is linked to the proteolysis tag, the module specifically binds to a target nucleotide sequence in a double stranded DNA, and the tag consists of (i) a peptide containing 3 hydrophobic amino acid residues at the C-terminal, or (ii) a peptide containing 3 amino acid residues at the C-terminal wherein at least a part of the amino acid residues is substituted by serine.

The present invention is related to the field of gene editing. In particular, the gene editing is directed toward single nucleotide base editing. For example, such single nucleotide base editing results in a conversion of a mutated base pair to a wild type base pair. The high accuracy and precision of the presently disclosed single nucleotide base gene editor is accomplished by a fusion protein including an NmeCas9 nuclease and an inlaid nucleotide deaminase protein domain. The Nme2Cas9 protospacer interacting domain may be replaced with an SmuCas9 protospacer interacting domain.

Methods and compositions to minimize barcode exchange during the preparation of barcoded next-generation sequencing libraries prepared from a single cell. The methods utilize oligonucleotides containing a 3-terminated blocking group or sequences that prevent amplification or extension.

The present invention pertains to a novel method for the generation of highly diverse RNA expressing vectors and vector libraries for use in targeted gene knock out, knock down and genome modification approaches. The invention pertains to a method for generating such higher order libraries without the need of classical cloning technologies. This is particularly useful for libraries based on large vectors wherein a sequence cannot be easily mutated with classical mutagenesis methods. The vectors and libraries generated according to the methods of the invention are in particular for RNA assisted silencing technologies such as RNA interference, and for targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system or similar RNA/DNA-encoded gene perturbation systems which use small guide RNAs to target the CRISPR complex to a specific genomic sequence. The invention provides also kits comprising the materials for performing the methods of the invention.

The present invention relates to improved semi-automated methods that permit the extraction of nucleic acids from samples, preparation of PCR and post-PCR preparation steps of DNA- libraries for next-generation sequencings methods that can be conducted. The methods and additional aspects relating to such methods are less laborious, safe costs, reagents and are less prone to contamination than comparable methods that are not automated.

Methods and compositions to minimize barcode exchange during the preparation of barcoded next-generation sequencing libraries prepared from a single cell. The methods utilize oligonucleotides containing a 3-terminated blocking group or sequences that prevent amplification or extension.

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

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5 or 3 single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3 ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5 ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5 or 3 single-strand protruding ends, along with any methylation present.