Engineered retrons and methods of use
Inventors
LADHA, Alim • Presnyak, Vladimir • Shcherbakova, Inna • Goodman, Brian • Mestre, Mario Rodriguez • Quinlan, Devin Scott • Jayaraman, Muthusamy • Scully, Stephen
Assignees
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Publication Number
US-11866728-B2
Publication Date
2024-01-09
Expiration Date
Abstract
Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g., mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivo to the host (e.g., mammalian) cell.
Core Innovation
The invention relates to a gene editing system comprising one or more delivery vehicles carrying RNA cargo for RNA-guided genome editing. The RNA cargo includes at least one mRNA molecule encoding a nucleic acid programmable nuclease and a retron reverse transcriptase, an engineered retron ncRNA, and a guide RNA for the nucleic acid programmable nuclease. The delivery vehicles deliver the nucleic acid programmable nuclease mRNA, the retron reverse transcriptase mRNA, the engineered retron ncRNA, and the guide RNA together across one or more delivery vehicles.
The engineered retron ncRNA comprises an HDR nucleotide sequence substituted into a retron ncRNA, and the HDR nucleotide sequence is substituted at a hairpin loop of the retron ncRNA. The retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to any one of SEQ ID NO:15833, SEQ ID NO:18547, or SEQ ID NO:19053, and the retron reverse transcriptase and the retron ncRNA are from the same phylogenetic clade.
The disclosure also describes recombinant retron-based genome editing using engineered retrons with retron RT, retron ncRNA, and guide RNA used with CRISPR/Cas9, including Cas9-RT fusion and configurations linking or fusing ncRNA with sgRNA or providing separate RNAs. The engineered retrons use RT-produced msDNA donor and HDR/repair to enable precise genome edits, and the document also includes molecular recording using engineered retrons to generate synthetic CRISPR protospacers for Cas1-Cas2 integration into CRISPR arrays.
Claims Coverage
The independent claims center on RNA cargo delivery vehicles that co-localize a nucleic acid programmable nuclease with a retron reverse transcriptase and an engineered retron ncRNA containing an HDR nucleotide sequence. The claim coverage includes five inventive features that refine the HDR substitution location, sequence identity constraints, phylogenetic clade relationship, and delivery across one or more vehicles.
RNA cargo delivery of nuclease, retron reverse transcriptase, engineered retron ncRNA, and guide RNA
The delivery vehicle(s) comprise RNA cargo including at least one mRNA encoding a nucleic acid programmable nuclease and a retron reverse transcriptase, an engineered retron ncRNA, and a guide RNA for the nucleic acid programmable nuclease.
Engineered retron ncRNA with HDR nucleotide sequence substituted into a hairpin loop
The engineered retron ncRNA comprises an HDR nucleotide sequence substituted into a retron ncRNA, wherein the HDR nucleotide sequence is substituted at a hairpin loop of the retron ncRNA.
Engineered retron ncRNA nucleotide identity to specified SEQ IDs
The retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to any one of SEQ ID NO:15833, SEQ ID NO:18547, or SEQ ID NO:19053.
Phylogenetic clade pairing of retron reverse transcriptase and engineered retron ncRNA
The retron reverse transcriptase and the retron ncRNA are from the same phylogenetic clade.
RNA components delivered across one or more delivery vehicles
Each delivery vehicle contains one or more of the RNA components, whereby one delivery vehicle or more than one delivery vehicle delivers the mRNA encoding the nucleic acid programmable nuclease and the retron reverse transcriptase, the engineered retron ncRNA, and the guide RNA.
Overall, the claim coverage centers on an RNA-cargo gene editing system that combines a nucleic acid programmable nuclease and guide RNA with a retron reverse transcriptase and an engineered retron ncRNA carrying an HDR nucleotide sequence inserted at a hairpin loop. The engineered ncRNA is constrained by sequence identity to specified SEQ IDs and is paired with a retron reverse transcriptase from the same phylogenetic clade, with delivery components distributed across one or more delivery vehicles.
Stated Advantages
Improve msDNA/donor production.
Reduce ncRNA immunogenicity.
Modulate msDNA function.
Increase targeted editing efficiency.
Documented Applications
Recombinant retron-based genome editing using engineered retrons with CRISPR/Cas9 to perform precise genome edits via RT-produced msDNA donor and HDR/repair.
Using engineered retrons to generate synthetic CRISPR protospacers for Cas1-Cas2 integration into CRISPR arrays, enabling molecular recording at the CRISPR-array level.
Genetically modifying a cell using an RNA cargo gene editing system that cuts a target sequence, generates RT DNA containing a donor polynucleotide, and integrates the donor polynucleotide at the target sequence.
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