Provided herein are, inter alia, compositions and methods for manipulation of genomes of living organisms.

The present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

Methods of enhancing homeostatic capacity in a subject are provided. Aspects of the methods include increasing the amplitude of an input resultant response of a homeostatic system component of the subject in a manner sufficient to enhance homeostatic capacity of the subject. Also provided are devices configured for use in practicing the methods. Aspects of the invention further include methods of treating a subject for a condition via enhancement of homeostatic capacity. The methods and devices described herein find use in a variety of applications.

Described herein are recombinant fermenting organisms having a heterologous polynucleotide encoding a phospholipase. Also described are processes for producing a fermentation product, such as ethanol, from starch or cellulosic-containing material with the recombinant fermenting organisms.

The invention is in the field of regulation of enzymatic activity in nucleic acid modifying reactions. It describes a method of regulating enzymatic activity by adding chelating agents to the reaction composition and exploits the fact that both the binding of divalent cations to these chelating agents and the pH of commonly used buffers is temperature dependent. PCR experiments that are hampered by non-specific side products can be regulated such that the target sequence is amplified in a more specific manner.

Nutritional formulas comprising long-chain polyunsaturated fatty acids (LC-PUFAs) are provided, along with methods and devices for preparing and/or administering nutritional formulas. In some embodiments, a percentage of the LC-PUFAs in the nutritional formula are in the form of monoglycerides and/or free fatty acids. In some embodiments, the nutritional formulas do not comprise added lipase. Also provided are methods for providing nutrition to a subject, methods for improving fat absorption, methods for improving cognitive ability, methods for preventing chronic lung disease, and methods for reducing the length of time a patient requires total parenteral nutrition.