Systems, methods and composition of using RNase III mutants to produce sRNA to control host pathogen infection
Inventors
Yin, Guohua • Lebrun, Erick Scott • Travers, Timothy S.
Assignees
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Publication Number
US-11800872-B2
Publication Date
2023-10-31
Expiration Date
Abstract
The current invention includes systems, methods and compositions for the generation of sRNA molecules using select RNase III mutants. In one preferred embodiment, invention includes systems, methods and compositions for the generation of sRNA molecules using RNase III mutants to control a host pathogen through the production and diffusion of sRNA molecules that may initiate an RNAi pathway response directed to a host pathogen. Additional embodiments of the current invention include systems, methods and compositions for the DICER-independent generation of sRNA molecules using select RNase III mutants.
Core Innovation
The invention relates to RNase III point mutants adapted for enhanced generation of small RNA (sRNA) from catalytic cutting of double stranded RNA (dsRNA). A genetically modified cell expresses a heterologous polynucleotide sequence operably linked to a promoter encoding an RNase III mutant, including an E38A-R107A-R108A mutant or a homologous RNase III mutant thereof including conserved residues E38, R107 and R108.
The mutant is associated with changes in dsRNA cleavage output toward discrete sRNA products, including 22-23 nucleotides. Such residue-mutant combinations can change dsRNA cleavage output to discrete sRNA sizes and can be used to enhance generation of sRNA that initiates an RNAi response pathway in a target host, independently of DICER.
Compositions are also described that comprise the E38A-R107A-R108A RNase III mutant from E. coli or Enterobacter, with specific residue substitutions. The described embodiments include co-expression of dsRNA targeting an essential pathogen gene, including an essential viral pathogen gene, to produce sRNA through catalytic cutting of dsRNA.
Claims Coverage
The independent claims cover five inventive features centered on using the E38A-R107A-R108A RNase III mutant to enhance catalytic cutting of dsRNA into sRNA, including compositions and sequence-selected variants.
Enhanced sRNA generation from dsRNA using E38A-R107A-R108A RNase III mutant
A genetically modified cell expressing a heterologous polynucleotide operably linked to a promoter sequence encoding an RNase III mutant from E. coli or Enterobacter adapted for enhanced generation of sRNA from catalytic cutting of dsRNA, wherein the RNase III mutant comprises an E38A-R107A-R108A RNase III mutant or a homologous RNase III mutant including conserved residues E38, R107 and R108.
Discretely sized dsRNA cleavage products for sRNA generation
A genetically modified cell wherein the E38A-R107A-R108A RNase III mutant preferentially cuts dsRNA into discrete sizes of 22 and 23 nucleotides (nt).
DICER-independent initiation of an RNAi response pathway
A genetically modified cell in which the RNAi response pathway in a target host is initiated independently of the DICER enzyme.
Composition of E38A-R107A-R108A RNase III mutant with specified residue substitutions
A composition comprising an E38A-R107A-R108A RNase III mutant from E. coli or Enterobacter, wherein a glutamic acid is replaced with an alanine at residue 38, and an arginine is replaced with an alanine at residues 107 and 108.
SEQ ID NO-selected polynucleotide and polypeptide variants encoding the mutant
Compositions comprising polynucleotide or polypeptide sequence variants selected from SEQ ID NO. 7, 11, 15, 12, 16, or 8, or a fragment thereof.
Across the independent claims, the inventive coverage centers on the E38A-R107A-R108A (or homologous) RNase III mutant for enhanced catalytic cutting of dsRNA into sRNA, including discrete 22-23 nt products, DICER-independent RNAi initiation, and compositions or constructs defined by specific SEQ ID NO-selected variants.
Stated Advantages
Enhanced generation of small RNA (sRNA) from catalytic cutting of double stranded RNA (dsRNA).
Generation of discrete dsRNA cleavage products corresponding to 22-23 nucleotides (nt) sRNA sizes.
RNAi response pathway initiation in a target host independently of DICER.
Documented Applications
Co-expression in a target host with dsRNA targeting an essential pathogen gene, including an essential viral pathogen gene, to initiate an RNAi response pathway using sRNA generated by catalytic cutting of dsRNA.
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