The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

A combination of virus vectors for genome editing is formed by arranging a polynucleotide encoding a split genome editing enzyme in each of a Tobamovirus vector and a Potexvirus vector and arranging a polynucleotide encoding a guide RNA in one of the vectors. It is found that when these virus vectors are introduced into a plant cell, a complex of a functional Cas9 protein and the guide RNA is formed in the plant cell, and a genome is edited in a target site-specific manner.

A combination of virus vectors for genome editing is formed by arranging a polynucleotide encoding a split genome editing enzyme in each of a Tobamovirus vector and a Potexvirus vector and arranging a polynucleotide encoding a guide RNA in one of the vectors. It is found that when these virus vectors are introduced into a plant cell, a complex of a functional Cas9 protein and the guide RNA is formed in the plant cell, and a genome is edited in a target site-specific manner.

A CRISPR-Cas3 system was successfully established in a eukaryotic cell.

A CRISPR-Cas3 system was successfully established in a eukaryotic cell.

A CRISPR-Cas3 system was successfully established in a eukaryotic cell.

A CRISPR-Cas3 system was successfully established in a eukaryotic cell.

Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.

The present invention relates to a method for introducing a polynucleotide into non-adhesively growing plant cells, comprising the following steps: providing a solid support having immobilized thereto the polynucleotide in dry state; contacting the plant cells with the polynucleotide on the solid support so as to obtain transformed plant cells; and optionally washing the plant cells.

The present invention relates to a method for introducing a polynucleotide into non-adhesively growing plant cells, comprising the following steps: providing a solid support having immobilized thereto the polynucleotide in dry state; contacting the plant cells with the polynucleotide on the solid support so as to obtain transformed plant cells; and optionally washing the plant cells.