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.

Disclosed herein is a modified transposon end sequence comprising a mosaic end sequence, wherein the mosaic end sequence comprises one or more mutation as compared to a wild-type mosaic end sequence, wherein the mutation comprises a substitution with a uracil, an inosine, a ribose, an 8-oxoguanine, a thymine glycol, a modified purine, or a modified pyrimidine. Also disclosed are transposome complexes comprising these modified transposon end sequences and methods of library preparation using these modified transposon end sequences.

A method includes incubation of an amplification mixture containing uracil-DNA glycosy lase enzyme (UNG enzyme), a deoxyribonucleotide triphosphate (dNTP) mixture, a DNA polymerase, a template including a sequence of interest, and at least one primer pair, inactivation of UNG enzyme in the amplification mixture, and amplification of a sequence of interest to form an in vitro transcription template. The in vitro transcription template may be used to produce a therapeutic polynucleotide.

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.

Methods for in situ amplification (ISA) of cfNA, such as cfDNA, in a sample are provided wherein the cfNA in the sample is not subject to a nucleic acid purification step. The methods disclosed may be used to generate an analyzable pool of cfNA present in the sample. The analyzable pool may be used with a variety of analytical techniques to characterize the nucleic acid in the sample. Methods of diagnosis, determining a therapeutic intervention and monitoring of a subject are also provided.

Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.

Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.

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.

Provided are a composition for inducing DNA single strand breaks in DNA, the composition comprising a cytidine deaminase, an inactivated target-specific endonuclease, and a guide RNA, a method for inducing a single-strand break in DNA, using the same, a method for analyzing a nucleic acid sequence of a base-editing-introduced DNA, and a method for identifying (or measuring or detecting) a base-editing site, base-editing efficiency at an on-target site, an off-target site, and/or target specificity.

Materials and methods for using modified Cas9-APOBEC fusion polypeptides for targeted modification of specific DNA sequences are provided herein.