This disclosure provides systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. PASTE combines gene editing technologies and integrase technologies to achieve unidirectional incorporation of genes in a genome for the treatment of diseases and diagnosis of disease.

The present invention provides DNA polymerases that are highly resistant to inhibitors, and that can shorten the entire nucleic acid amplification reaction time by shortening the reverse transcription reaction time in a nucleic acid amplification method, in particular, in PCR or RT-PCR. The DNA polymerase is characterized by having reverse transcription activity, and comprising at least one amino acid modification at position 509 or 744 in SEQ ID NO: 1 or 2. In particular, the amino acid modification at position 509 or 744 in SEQ ID NO: 1 or 2 is substitution with histidine, lysine, or arginine.

The present invention provides novel engineered reverse transcriptase enzymes that afford beneficial improvements in thermal stability, processivity, cDNA yields and elimination of secondary enzymatic activity. The present invention also provides methods for amplifying template nucleic acids using such reverse transcriptase enzymes. This invention addresses deficiencies in the current state of the art reverse transcriptase enzymes in RNA detection and analysis including deficiencies in detection sensitivity, specificity, side enzyme activities, enzyme stability and synthesis capacity, especially when using template nucleic acids ranging in length, secondary structure and nucleotide content.

The present invention relates to a chimeric enzyme comprising a CRISPR class 2 type II enzyme backbone, wherein the HNH domain in the backbone has been replaced, essentially, by a peptide or protein domain having catalytic activity on a single stranded polynucleotide.

The lipid nanoparticle compositions provided herein preferentially deliver and/or transfect the lung. Also provided herein are therapeutic polynucleotides, e.g. TERT mRNA, which may be delivered with the LNP formulations for the treatment of lung disease and fibrosis.

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.

Engineered retrons, modified to enhance production of multicopy single-stranded DNA (msDNA), are provided. In addition, vector systems encoding such engineered retrons and methods of using engineered retrons and vector systems encoding them in various applications such as CRISPR/Cas-mediated genome editing, recombineering, cellular barcoding, and molecular recording are also disclosed.

The subject application pertains to compositions and methods for enhancing reverse transcriptase (RT) activity and/or reducing the inhibition of RT by inhibitors, such as formalin, tannic acid and/or heparin. In some embodiments, RT inhibition is reduced by the addition of potassium glutamate, histidine hydrochloride monohydrate, poloxamer 188, or any combination thereof to a reaction mixture comprising a polymerase. In other embodiments, RT is enhanced through the addition of a polyvinyl sulfonic acid sodium salt (PVSA) to a reaction mixture. The subject application also provides oligonucleotide primers for use in the reverse transcription of target sequences and its enhancement. These primers have high GC content or low GC content. Methods of using a RT inhibition reducer or a RT enhancer in a composition with an RNA template and RT improves RT yield, RT sensitivity, or RT tolerance to various chemicals are also provided.

Provided herein are methods and systems for altering the genome of an organelle. In some embodiments, the method comprises introducing into an organelle a recombinant DNA construct comprising a first polynucleotide encoding at least one guide RNA and a second polynucleotide encoding a polynucleotide guided polypeptide; and growing a cell comprising the organelle under conditions in which the first polynucleotide and the second polynucleotide are each expressed.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.