Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated virus (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Recombinant AAV vectors and particles for the delivery of the split Cas9 protein or nucleobase editor, and methods of using such AAV vectors and particles are also provided.

Provided herein are methods of identifying a methylation status of an analyte in a biological sample. Also provided herein are methods that combine identifying the methylation status with spatial technology to identify the location of a methylation status in a biological sample.

Provided herein are methods of identifying a methylation status of an analyte in a biological sample. Also provided herein are methods that combine identifying the methylation status with spatial technology to identify the location of a methylation status in a biological sample.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Methods and compositions for performing highly sensitive in vitro assays to define substrate preferences and off-target sites of nucleic-acid binding, modifying, and cleaving agents.

Methods and compositions for performing highly sensitive in vitro assays to define substrate preferences and off-target sites of nucleic-acid binding, modifying, and cleaving agents.

Described herein is a multiplex 5mC marker barcode counting (MMBC) to quantify 5mC markers in DNA, in which multiple 5mC markers could be targeted for capture and linear amplification. The inventors' method provides highly sensitive and specific quantification of multiple 5mC loci. Accordingly, aspects of the disclosure relate to a method for detecting methylated or unmethylated cytosines in one or more regions of target nucleic acids, the method comprising i) combining a solution comprising the target nucleic acids with a deaminating agent to convert unmethylated cytosines in the target nucleic acids to uracils; ii) next contacting the solution with at least two probes under conditions that allow for the hybridization of the two probes to one target nucleic acid region; wherein a terminal end from each probe hybridizes adjacently to the target nucleic acid region; iii) contacting the solution comprising the hybridized probes and target nucleic acids with a ligase under conditions that allow for the ligation of the terminal ends of the adjacently hybridized probes; and iv) detecting the adjacently hybridized ligated probes in the solution.