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Method for improving plant genetic transformation and gene editing efficiency — Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences (US12416013B2)

Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US12416013B2, Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences, Caixia GAO, en, 2025

ABSTRACT

Abstract

The present invention provides a method for improving plant genetic transformation and gene editing efficiency. Specifically, the method improves the regeneration efficiency of plant genetic transformation and/or improves the efficiency of plant gene editing by expressing genes that promote plant cell division, especially meristematic cell division.

Description

REFERENCE TO SEQUENCE LISTING TEXT FILE

The Sequence Listing text file associated with the instant disclosure has been electronically submitted to the United States Patent and Trademark Office via EFS as a 123.118 byte UTF-8-encoded text file created on Jul. 5, 2023 and entitled â1547_48_PCT_US_ST25.xmlâ. The Sequence Listing submitted via EFS is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to a method for improving plant genetic transformation and gene editing efficiency. More specifically, the present invention relates to improving regeneration efficiency of plant genetic transformation and/or improving efficiency of plant gene editing by expression of genes which can promote division of plant cells, especially meristematic cells.

BACKGROUND OF THE INVENTION

Development of crop genetic breeding includes artificial selection breeding, cross breeding, mutation breeding and molecular marker assisted breeding using molecular technology. As the variety genetic diversity is gradually reduced, the bottleneck effect of traditional breeding is more and more obvious: it is difficult to use conventional breeding technology to obtain breakthrough new varieties to meet the requirement of human and sustainable agricultural development. The rapid development of life sciences makes it possible to enter the post-genome era from the âreadâ phase of the biological genetic information, and the accurate ârewritingâ of the genome and the ânew designâ are becoming reality. This kind of biological technical means designed for creating new trait or living body shows great prospect in the field of disease treatment, medicine, manufacturing, especially agriculture and so on.

Genome editing technology is a revolutionary technical means appearing in the current life science which can realize accurate, efficient and specific rewriting of the genome and has revolutionary pushing effect to the research and exploration of life science. Gene editing refers to deleting, replacing, or inserting operation of the target gene so as to modify the genetic information to obtain a new function or phenotype, even creating a new species. Development of efficient and accurate breeding technology suitable for crops using gene editing technology will break the defect of the traditional breeding, realizing molecular design breeding of precise transformation from the genome. It has important strategic significance for the development of future agriculture.

Current gene editing technology mainly comprises ZFN. TALEN and CRISPR/Cas system. CRISPR/Cas system, due to its high efficiency and flexibility, is currently the simplest and widely used gene editing technology system. In CRISPR/Cas system, Cas protein can target any position in the genome under the guide action of the artificial designed guide RNA (guide RNA). Base editing system is a new gene editing technology developed based on CRISPR system, which can be divided into cytosine base editing system and adenine base editing system, wherein the respectively deaminase and adenine deaminase are fused with Cas9 single-chain nickase. Under the targeting function of the guide RNA, Cas9 single-chain nickase generates a single-stranded DNA region, so the deaminase can efficiently respectively remove amino group of the C and A nucleotide on the single-stranded DNA of the targeting position, resulting in U base and I base, which can be repaired as T base and G base by the repair process of the cell itself. Base editing technology overcomes the defect of traditional DSB-mediated gene editing, which can efficiently realize the precise replacement of a single base. CRISPR/Cas system-mediated robust genetic engineering technology system will provide strong technical support for gene research and new plant molecular design breeding, and will accelerate the cultivation of new variety of crops and realize sustainable development of agriculture.

A key step of plant gene editing is to deliver the gene editing nuclease protein or coding nucleic acid to the plant cell to realize the editing of the target gene. The present plant genome editing delivery technology is mainly realized by genetic transformation and tissue culture technology, mainly comprising agrobacterium mediated method and gene gun method. Important progress has been made in plant transformation over the past few years, but transformation of many agronomically important plants (e.g., maize, soybeans, canola, wheat, indica rice, sugar cane and sorghum; and inbred lines) is still difficult and time-consuming. Generally, the only method causing the culture reaction is to optimize the culture medium components and/or explant materials and sources, which results in the success in some gene type, but many important crop plants (including excellent inbred line or variety) do not generate beneficial culture reaction and regeneration technology system. Although the transformation of a pattern genotype may be effective, the process of gradually introgressing the transgene into the product inbred line is laborious, expensive and time-consuming. Especially for monocotyledon wheat, efficiency of current gene gun and agrobacterium transformation method is low and greatly limited by the genotype, and a long-term tissue culture process is required. At present, the maximum bottleneck of wheat gene editing is the low efficiency of the current traditional wheat transformation system, the large technical difficulty, limitation by genotype and low throughput.

In order to facilitate the research of plant gene function and molecular design breeding more efficiently utilizing gene editing technology, establishing and digging a method to improve the plant transformation efficiency and shorten the time of tissue culture has important meaning.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for improving plant cell regeneration efficiency in plant transformation, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF nd an expression construct comprising a coding nucleic acid sequence of GIF;

(b) regenerating an intact plant from the plant cell.

In another aspect, the present invention provides a method for improving the transformation efficiency of an exogenous nucleic acid sequence of interest in a plant or for transforming an exogenous nucleic acid sequence of interest into a plant, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF nd an expression construct comprising a coding nucleic acid sequence of GIF;

(b) introducing at least one expression construct comprising at least one exogenous nucleic acid sequence of interest into the plant cell; and (c) regenerating an intact plant from the plant cell.

In another aspect, the present invention provides a method for improving gene editing efficiency in a plant or for gene editing in a plant, the method comprising:

(a) introducing into a plant cell

i) an expression construct comprising a coding sequence of WUS, an expression construct comprising a coding sequence of BBM and an expression construct comprising a coding sequence of SERK; and/or ii) an expression construct comprising a coding sequence of GRF and an expression construct comprising a coding sequence of GIF;

(b) introducing at least one expression construct comprising at least one exogenous sequence of interest to the plant cell, wherein the at least one exogenous sequence of interest encodes a component of a gene editing system; or introducing at least one component of a gene editing system to the plant cell; and (c) regenerating an intact plant from the plant cell.

The invention further provides a kit for carrying out the method of the invention, comprising at least i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF.

The present invention also provides use of i) an expression construct comprising a coding nucleic acid sequence of WUS; an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF, for improving plant cell regeneration efficiency in plant transformation, for improving the transformation efficiency of exogenous nucleic acid sequence of interest in the plant or for improving the gene editing efficiency in the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Expression constructs for detecting the influence of a plurality of DR combinations on genetic transformation and gene editing efficiency.

FIG. 2 shows expression constructs for verifying the effect of GRF4 and gif1 combination on genetic transformation and gene editing efficiency in wheat.

FIG. 3 shows the expression constructs for GRF4 and gif1 combinatorial optimization experiment.

FIG. 4 shows the soybean GRF/gif expression construct.

FIGS. 5 A- 5 K . The effect of GmGRF-GmGIF1 complexes on regeneration, transformation, and genome editing of the soybean cultivar Williams 82. ( FIG. 5 A ) Schematic representations of T-DNA regions each containing one of the four GmGRF-GmGIF1 complexes. pBSE401 is the CRISPR/Cas9 control construct. ( FIG. 5 B ) Schematic of the GmFAD2 sgRNA target sites in the two subgenomes. ( FIG. 5 C ) The general procedure for Agrobacterium -mediated transformation of soybean. ( FIG. 5 D ) Comparison of the effects of four GmGRF-GmGIF1 complexes on regeneration frequencies of Williams 82. RF (regeneration frequency)=no. of explants with multiple buds/explant numberÃ100%. ( FIG. 5 E ) Numbers of putative glufosinate-resistant elongated shoots â¥2 cm in length (on day 50 after transformation) and shoots â¥9 cm in length (on day 75 after transformation) regenerated from explants transformed with the four GmGRF-GmGIF1 complexes and pBSE401, respectively. ( FIG. 5 F ) Average numbers of elongated shoots regenerated from single explants transformed with the four GmGRF-GmGIF1 complexes and pBSE401, respectively. ( FIG. 5 G ) Regeneration of explants transformed with pGmGRF5-GmGIF1 and pBSE401 and cultured in medium supplemented with 5.0 mg/L glufosinate on day 50 after infection. ( FIG. 5 H ) Typical explants with regenerated shoots cultured in medium supplemented with 5.0 mg/L glufosinate on day 75 after infection with pGmGRF5-GmGIF1 and pBSE401, respectively. ( FIG. 5 I ) Transformation efficiencies of Williams 82 transformed with the four GmGRF-GmGIF1 complexes and pBSE401, respectively. ( FIG. 5 J ) GmFAD2 mutation frequencies with CRISPR/Cas9 treated by four pGmGRF-GmGIF1 constructs and pBSE401, respectively. ( FIG. 5 K ) Performance of mature GmFAD2-edited soybean plants (160 days after transformation) transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. All values and error bars are mean valuesÂ±s.e.m. of three independent experiments.

FIGS. 6 A- 6 G . The effect of the GmGRF5-GmGIF1 complex on regeneration, transformation, and genome editing of two soybean cultivars Zhonghuang 13 and Hefeng 25. ( FIG. 6 A ) Regeneration frequencies of explants transformed with pGmGRF5-GmGIF1 and pBSE401 on day 50 after transformation. ( FIGS. 6 B and 6 C ) Numbers of putative glufosinate-resistant elongated shoots (>2 cm in length) on day 50 after transformation and elongated shoots (>9 cm in length) on day 75 regenerated from explants of Zhonghuang 13 ( FIG. 6 B ) and Hefeng 25 ( FIG. 6 C ) transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. ( FIG. 6 D ) Shoot induction, shoot proliferation and shoot elongation in Hefeng 25 explants transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. ( FIG. 6 E ) Average numbers of elongated shoots regenerated from single explants of Zhonghuang 13 and Hefeng 25 transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. ( FIG. 6 F ) Transformation efficiencies of Zhonghuang 13 and Hefeng 25 transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. ( FIG. 6 G ) GmFAD2 mutation rates in Zhonghuang 13 and Hefeng 25 transformed with pGmGRF5-GmGIF1 and pBSE401, respectively. All values and error bars are mean valuesÂ±s.e.m. of three independent experiments.

FIG. 7 A- 7 D . The effect of the GmGRF5-GmGIF1 complex on regeneration, transformation, and genome editing efficiencies in strawberry cultivar Benihoppe. ( FIG. 7 A ) Schematic representation of the T-DNA region containing the GmGRF5-GmGIF1 complex. pHUE411-GFP is the CRISPR/Cas9 control construct. ( FIG. 7 B ) Effects of the GmGRF5-GmGIF1 complex on strawberry regeneration, transformation, and genome editing efficiencies. The data were collected on day 35 after explants were transformed with pHUE411-GFP-GmGRF5-GmGIF1 and pHUE411-GFP. RF (regeneration frequency)=no. of regenerated shoots/total explantsÃ100%. TE (transformation efficiency)=no. of transgenic shoots/total explantsÃ100%. MF (mutation frequency)=no. of mutants/total explantsÃ100%. ( FIG. 7 C ) GFP positive calli from explants transformed with pHUE411-GFP-GmGRF5-GmGIF1 and pHUE411-GFP, on day 21 after transformation. ( FIG. 7 D ) Calli and shoots from explants transformed with pHUE411-GFP-GmGRF5-GmGIF1 and pHUE411-GFP, respectively, on day 35 after transformation.

REFERENCE TO SEQUENCE LISTING TEXT FILE

TECHNICAL FIELD

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for improving plant cell regeneration efficiency in plant transformation, the method comprising:

(a) introducing into a cell of the plant

(b) regenerating an intact plant from the plant cell.

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF nd an expression construct comprising a coding nucleic acid sequence of GIF;

(b) introducing at least one expression construct comprising at least one exogenous nucleic acid sequence of interest into the plant cell; and (c) regenerating an intact plant from the plant cell.

In another aspect, the present invention provides a method for improving gene editing efficiency in a plant or for gene editing in a plant, the method comprising:

(a) introducing into a plant cell

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Expression constructs for detecting the influence of a plurality of DR combinations on genetic transformation and gene editing efficiency.

FIG. 2 shows expression constructs for verifying the effect of GRF4 and gif1 combination on genetic transformation and gene editing efficiency in wheat.

FIG. 3 shows the expression constructs for GRF4 and gif1 combinatorial optimization experiment.

FIG. 4 shows the soybean GRF/gif expression construct.

FIGS. 8 A- 8 H . Comparison of the effects of TaGRF4-TaGIF1 and mTaGRF4-TaGIF1 on regeneration and genome editing in two common wheat cultivars Kenong 199 and Bobwhite. ( FIG. 8 A ) Schematic representation of common wheat GIF1, GRF4, and mutated GRF4. The dotted lines indicate the interaction between the SNH and QLQ domains. mTaGRF4 was created by introducing five point mutations in the miRNA396 target site of common wheat TaGRF4. ( FIG. 8 B ) Schematic representations of constructs pTaGRF4-TaGIF1, pmTaGRF4-TaGIF1, and the base editor, pUBI-A3A. pUBI-GFP is the control construct. ( FIG. 8 C ) General procedure for transgene-free genome editing in common wheat by transient expression of a cytosine base editor. ( FIG. 8 D ) Comparison of the effects of TaGRF4-TaGIF1 and mTaGRF4-TaGIF1 on regeneration frequencies of Bobwhite and Kenong199. RF (regeneration frequency)=no. of regenerated shoots/immature embryos bombardedÃ100%. ( FIG. 8 E ) Comparison of the effects of TaGRF4-TaGIF1 and mTaGRF4-TaGIF1 on genome editing frequencies in Bobwhite and Kenong199. MF (mutation frequency)=no. of mutants/immature embryos bombardedÃ100%. ( FIG. 8 F ) Primer sets for detecting transgene-free mutants, and the outcome of tests on 22 representative TaALS mutant plants (Kenong 199). ( FIG. 8 G ) The Transgene-free frequencies in Bobwhite and Kenong199 wheat cultivars transformed with TaGRF4-TaGIF1, mTaGRF4-TaGIF1 and pUBI-GFP (control construct), respectively. ( FIG. 8 H ) Transgene-free mutant plants regenerated from Kenong 199 immature embryos transiently-expressing mTaGRF4-TaGIF1 and the cytosine base editor A3A-PBE, do not exhibit abnormal growth. In ( FIG. 8 D ), ( FIG. 8 E ) and ( FIG. 8 G ), values and error bars are meansÂ±s.e.m. of three independent experiments.

FIGS. 9 A- 9 C . The effect of transient expression of the mutated TaGRF4-TaGIF1 complex on common wheat regeneration and genome editing efficiencies in nine elite wheat cultivars. ( FIG. 9 A ) The regeneration frequencies of nine elite common wheat cultivars transformed with pmTaGRF4-TaGIF1 and pUBI-GFP (control construct). Values and error bars are meansÂ±s.e.m. of three independent experiments. ( FIG. 9 B ) Regenerated plants of Xiaoyan 54 and Zhongmai 175 transformed with mTaGRF4-TaGIF1 and pUBI-GFP (control construct), respectively, 28 days after transformation. ( FIG. 9 C ) The mutation frequencies in the nine elite common wheat cultivars transformed with pmTaGRF4-TaGIF1 and pUBI-GFP (control construct). MF (mutation frequency)=total no. of mutants/total immature embryos bombardedÃ100%.

FIGS. 10 A- 10 C . Detection of mutations and transgene-free mutant plants in 15 representative Williams 82 soybean lines transformed with pGmGRF5-GmGIF1. ( FIG. 10 A ) Mutations in the GmFAD2 gene from the 15 representative soybean lines identified by PCR-RE assays. Lanes 1 to 15 show digests of the PCR fragments amplified from the transgenic soybean plants using BstXI. Lanes labeled CK show the digests of PCR fragments amplified from a wild-type control plant. ( FIG. 10 B ) The outcome of tests for transgene-free mutants using two primer sets in 15 representative gmfad2 mutant plants. Lanes without a band indicate transgene-free mutants. Lanes labeled CK are the PCR fragments amplified from a WT plant. ( FIG. 10 C ). Sanger sequencing of the GmFAD2 gene in wild type and the edited gmfad2 mutants.

FIGS. 11 A- 11 C . Phylogenetic analysis of GRFs from Glycine max (Gm) and Fragaria vesca (Fve) and the sgRNA designed to generate mutant strawberry FaPL genes. ( FIG. 11 A ) Clustal W was used to align 31 GRFs, including nine FveGRFs and 22 GmGRFs. MEGA 7.0 was used to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replications. ( FIG. 11 B ) Schematic of the sgRNA designed to target the FaPL gene. ( FIG. 11 C ) Detection of mutations by Sanger sequencing in regenerated strawberry lines transformed with pHUE411-GFP-GmGRF5-GmGIF1.

FIGS. 12 A and 12 B . Multiple sequence alignment of the deduced GRF4 and GIF1 proteins, which are the most similar between common wheat and rice. ( FIG. 12 A ) The GRF4 from common wheat shows 62.5% conservation with the GRF4 from rice at the amino acid level. ( FIG. 12 B ) The GIF1 from common wheat shows 86.5% conservation with the GIF1 from rice at the amino acid level. Sequences were compared using Geneious Prime.

FIG. 13 . Schematic of the TaALS sgRNA target sites in the common wheat genome. SgRNA target sites within a conserved region of common wheat TaALS homeologs were targeted by the base editing systems. The EcoO109I restriction enzyme site in the sgRNA target sequence was used for mutation detection.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meaning commonly understood by those skilled in the art, and the protein and nucleic acid chemistry used herein, molecular biology, cell and tissue culture, microbiology, immunology related term and laboratory operation steps are widely used in the corresponding field and conventional steps. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those of skill in the art, and are more fully described in the following literature: Sambrook. J., Fritsch. E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter âSambrookâ). At the same time, for a better understanding of the present invention, the definitions and explanations of the related terms are provided below.

As used herein, the term âand/orâ encompasses all combinations of items connected by the term, and each combination should be regarded as individually listed herein. For example. âA and/or Bâ covers âAâ, âA and Bâ, and âBâ. For example, âA, B, and/or Câ covers âAâ, âBâ, âCâ, âA and Bâ, âA and Câ, âB and Câ, and âA and B and Câ.

When the term âcompriseâ is used herein to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotide at one or both ends of the protein or nucleic acid, but still have the activity described in this invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical conditions (for example, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide. Therefore, when describing the amino acid sequence of specific polypeptide in the specification and claims of the present application, although it may not include the methionine encoded by the start codon at the N-terminus, the sequence containing the methionine is also encompassed, correspondingly, its coding nucleotide sequence may also contain a start codon; vice versa.

âGenomeâ as used herein encompasses not only chromosomal DNA present in the nucleus, but also organelle DNA present in the subcellular components (e.g., mitochondria, plastids) of the cell.

The term âexogenousâ with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

âPolynucleotideâ, ânucleic acid sequenceâ, ânucleotide sequenceâ, or ânucleic acid fragmentâ are used interchangeably to refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5â²-monophosphate form) are referred to by their single letter designation as follows: âAâ for adenylate or deoxyadenylate (for RNA or DNA, respectively). âCâ for cytidylate or deoxycytidylate, âGâ for guanylate or deoxyguanylate, âUâ for uridylate. âTâ for deoxythymidylate, âRâ for purines (A or G). âYâ for pyrimidines (C or T), âKâ for G or T. âHâ for A or C or T. âIâ for inosine, and âNâ for any nucleotide.

âPolypeptideâ, âpeptideâ, âamino acid sequenceâ and âproteinâ are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms âpolypeptideâ, âpeptideâ, âamino acid sequenceâ, and âproteinâ are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

As used herein, an âexpression constructâ refers to a vector suitable for expression of a nucleotide sequence of interest in an organism, such as a recombinant vector. âExpressionâ refers to the production of a functional product. For example, the expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (such as transcribe to produce an mRNA or a functional RNA) and/or translation of RNA into a protein precursor or a mature protein.

âExpression constructâ of the invention may be a linear nucleic acid fragment (including a DNA or RNA fragment), a circular plasmid, a viral vector.

The âexpression constructâ of the present invention may comprise a regulatory sequence and a nucleic acid sequence of interest operably linked thereto. The regulatory sequence and the nucleic acid sequence of interest may be of different sources, or are of the same origin but are arranged in a manner different from that normally found in nature.

âRegulatory sequenceâ and âregulatory elementâ are used interchangeably and refer to a nucleotide sequence which is located upstream (5â² non-coding sequences), within, or downstream (3â² non-coding sequences) of a coding sequence, and influence the transcription, RNA processing or stability, or translation of the associated coding sequence. The regulatory sequence may include, but is not limited to, a promoter, a translation leader sequence, an intron and a polyadenylation recognition sequence. âPromoterâ refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment. In some embodiments of the invention, the promoter is a promoter capable of controlling gene transcription in a cell regardless of whether it is derived from the cell. The promoter may be a constitutive promoter or a tissue specific promoter or a developmentally regulated promoter or an inducible promoter.

As used herein, the term âoperably linkedâ refers to a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) and a nucleic acid sequence (e.g., a coding sequence or open reading frame), are linked such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

âIntroducingâ a nucleic acid molecule (e.g., an expression construct) into a plant cell refers to that the nucleic acid molecule is presented to the plant cell such that the nucleic acid molecule enter the interior of the plant cell.

âRegenerationâ refers to the process of growing an intact plant from one or more plant cells (e.g., plant protoplasts, callus or explants).

II. Improved Plant Transformation

In one aspect, the present invention provides a method for improving plant cell regeneration efficiency in plant transformation, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF;

(b) regenerating an intact plant from the plant cell.

In another aspect, the present invention provides a method for improving transformation efficiency of an exogenous nucleic acid sequence of interest in a plant or for transforming an exogenous nucleic acid sequence into a plant, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding sequence of SERK; and/or ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF;

(b) introducing at least one expression construct comprising at least one exogenous nucleic acid sequence of interest to the plant cell; and (c) regenerating an intact plant from the plant cell.

In some embodiments of this aspect, step (a) and step (b) are carried out at the same time. In some embodiments of this aspect, step (a) is performed prior to step (b). In some embodiments of this aspect, step (b) is performed prior to step (a). In some embodiments of this aspect, step (c) is performed after step (a) and step (b).

In another aspect, the present invention provides a method for improving gene editing efficiency in a plant or for gene editing in a plant, the method comprising:

(a) introducing into a cell of the plant

(b) introducing at least one expression construct comprising at least one exogenous sequence of interest to the plant cell, wherein the at least one exogenous sequence of interest encodes a component of a gene editing system; or introducing at least one component of the gene editing system to the plant cell; and (c) regenerating an intact plant from the plant cell.

The invention further provides a kit for carrying out the method of the invention, comprising at least i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK; and/or ii) an expression construct comprising a coding sequence of GRF and an expression construct comprising a coding sequence of GIF.

WUS (WUSCHEL), BBM (BABY BOOM) and SERK (Somatic Allogenesis receptor-like kinase) are conservative development regulatory factors (DR) widely present in plants. The inventors have surprisingly found that co-expression of the combination of WUS. BBM and SERK in the plant cell can significantly improve the efficiency of the plant cell to regenerate into intact plants, and significantly improve transformation efficiency of an exogenous nucleic acid sequence of interest into the plant. When the exogenous nucleic acid sequence of interest encodes a gene editing system, the gene editing efficiency can be significantly improved.

Examples of WUS, BBM and SERK suitable for use in the present invention include, but are not limited to, WUS, BBM, and SERK from Arabidopsis , canola, strawberry, potato, rice, tomato, soybean, corn or wheat.

In some embodiments of various aspects of the invention. WUS is corn WUS (ZmWUS), BBM is the corn BBM (ZmBBM), or SERK is corn SERK (ZmSERK). In some embodiments, the ZmWUS comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the ZmBBM comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the ZmSERK comprises the amino acid sequence shown in SEQ ID NO: 3.

In some embodiments of various aspects of the present invention, at least two or at least three or all of the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM, the coding nucleic acid sequence of SERK, and the at least one exogenous nucleic acid sequence of interest are placed in a same expression construct. In some embodiments, the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM, the coding nucleic acid sequence of SERK and the at least one exogenous nucleic acid sequence of interest are respectively placed in different expression constructs.

In some embodiments of various aspects of the invention, the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM, and the coding nucleic acid sequence of SERK are placed in the same expression construct, while the at least one exogenous nucleic acid sequence of interest is placed in another expression construct.

In some embodiments of various aspects of the present invention, the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM, the coding nucleic acid sequence of SERK and/or the at least one exogenous nucleic acid sequence of interest are operatively connected to transcription regulatory elements.

Methods of expressing different proteins by the same expression construct are known in the art. For example, the different proteins can be placed in the same expression construct under the control of different transcriptional regulatory elements (e.g., different promoters). Alternatively, different proteins can be fused by self-cleaving peptide (e.g., 2A peptide, including but not limited to P2A, E2A; F2A and T2A, etc.), then expressed under the control of the same transcriptional control element (e.g., different promoter), so that separate different proteins can be generated by self-cleavage of the self-cleaving peptide after translation or translation. Alternatively, an internal ribosome entry site (IRES) can be inserted between the coding nucleic acid sequences of different proteins.

GRFs (Growth Factors) are specific transcription factors in plants, mainly controlling plant cell size, chloroplast proliferation, stamen development, osmotic stress and other plant growth and development processes. GRF transcription factors are widely existed in the plant, mainly comprising two conserved domains: QLQ and WRC. The QLQ domain of GRFs can interact with the SNH domain (SYT N-terminal domain) in GIF (GRF-interacting factor) proteins, so as to exercise transcriptional activation function. WRC domain comprises 1 functional nuclear localization signal and 1 DNA binding motif, plays a role in DNA binding. Generally, QLQ and WRC domain are located at the N terminal of the GRFs. However, some GRFs also have a second WRC domain at the C terminal.

âGIFâ (GRF-interacting factor) is a protein that can form transcription co-activation factor complexes with GRF. GIFs are homologous to human transcription co-activation factor synovial sarcoma transport protein (synovial translocation protein. SYT). In Arabidopsis thaliana , GIF plays a role in cell proliferation during blade development and maintains proliferation ability of the meristematic cells during flower organ development.

The inventors have further surprisingly found that co-expression of the combination of GRF and GIF in plant cell can significantly improve the regenerating efficiency of a plant cell into intact plant, and significantly improve the transformation efficiency of exogenous nucleic acid sequence of interest into the plant. When the exogenous nucleic acid sequence of interest encodes a gene editing system, the gene editing efficiency can be significantly improved.

Examples of GRFs suitable for use in the present invention include, but are not limited to, GRF from Arabidopsis , canola, potato, rice, tomato, soybean, corn or wheat. Examples of GIF suitable for use in the present invention include, but are not limited to, GIF from Arabidopsis , canola, potato, rice, tomato, soybean, corn or wheat. However, as long as it can form transcription co-activating factor complex, GRF and GIF in the present invention does not necessarily have the same origin.

In some embodiments of the invention, the GRF is wheat GRF. Suitable wheat GRFs include, but are not limited to, wheat GRF4. In some embodiments, the wheat GRF4 comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments of the invention, the GIF is wheat GIF. Suitable wheat GIFs include, but are not limited to, wheat GIF1. In some embodiments, the wheat GIF1 comprises the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments of the invention, the GRF is soybean GRF. Suitable soybean GRF include, but are not limited to, soybean GRF5, soybean GRF6, soybean GRF11, or soybean GRF11. In some embodiments, the soybean GRF5 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the soybean GRF6 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the soybean GRF11 comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the soybean GRF18 comprises the amino acid sequence of SEQ ID NO: 10.

In some embodiments of the invention, the GIF is derived from soybean GIF. Suitable soybean GIFs include, but are not limited to, soybean GIF1. In some embodiments, the soybean GIF1 comprises the amino acid sequence shown in SEQ ID NO: 11.

Many of the transcription factors in the plant including GRF are regulated by miRNAs. For example, GRF4 is negatively controlled by miR396. The invention surprisingly found that mutating the miRNA binding site in GRF can significantly improve the effect of the GRF/GIF combination in improving efficiency of plant cell regeneration and plant genetic transformation.

Therefore, in some embodiments of the present invention, the GRF comprises a mutated miRNA binding site, so as not to be regulated by the miRNA. Examples of the miRNAs include, but are not limited to, miR396, depending on the particular GRF. In some embodiments, the GRF with the mutated miRNA binding site comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments of the present invention, at least two or all of the coding sequence of the GRF, the coding sequence of the GIF and the at least one exogenous nucleic acid sequence of interest are placed in a same expression construct.

In some embodiments of the present invention, the coding sequence of the GRF and the coding sequence of GIF are placed in a same expression construct, while the at least one exogenous nucleic acid sequence of interest is placed in another expression construct.

In some embodiments of the present invention, the coding sequence of the GRF, the coding sequence of the GIF and the at least one exogenous nucleic acid sequence are operatively linked to transcription control sequences.

In some embodiments of the invention, the GRF is fused to the GIF. In some embodiments, the GRF is fused to the N terminal of the GIF. In some embodiments, the GRF is fused to the GIF through a linker. An exemplary linker comprises the sequence AAAA (SEQ ID NO: 12) or SGGS (SEQ ID NO: 13). Preferably, the linker is AAAA.

In some embodiments of the present invention, âan expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIFâ encompasses an expression construct comprising a coding sequence of the fusion protein of GRF and GIF.

In some embodiments of the present invention, the fusion protein of the GRF and GIF comprises the amino acid sequence encoded by any one of SEQ ID NO: 17-22. In some embodiments of the present invention, the fusion protein of the GRF and GIF is encoded by any one of SEQ ID NO: 17-22. In some embodiments of the present invention, the fusion protein of GRF and GIF comprises the amino acid sequence encoded by any one of SEQ ID NO: 23-28.

The âat least one exogenous nucleic acid sequence of interestâ may be any nucleic acid sequence to be transformed into the plant. For example, the exogenous nucleic acid sequence of interest can encode an agronomic trait, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and a trait important for the commercial product. The nucleic acid sequence of interest may also include those nucleic acid sequences involved in oil, starch, carbohydrate or nutrient metabolism, and those nucleic acid sequences affecting seed size, sucrose content, and the like.

In some preferred embodiments of the present invention, the at least one exogenous nucleic acid sequence encodes a component of a gene editing system, so as to carry out gene editing in the plant.

âGene editingâ, also known as genome editing, uses a sequence-specific nuclease or a derivative thereof for nucleotide insertion, deletion or substitution in the genome of an organism. Gene editing generally results in site-specific double-strand break (DSB) at the desired position in the genome, and then introducing desired DNA insertion, deletion or substitution by the process of repairing DSB. However, gene editing can also cover base editing technology, transcriptional activation or inhibition, epigenetic modification technology, which does not relate to DSB, as long as it has sequence specificity.

The gene editing system used is not particularly limited in the present invention. For example, a gene editing system suitable for use in the present invention includes but is not limited to zinc finger nuclease (ZFN), meganuclease (MGN); transcription activating factor-like effector nuclease (TALEN) and CRISPR (Clustered regularly interspaced short palindromic repeats) system. âZinc finger nucleasesâ are artificial restriction enzymes prepared by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger DNA binding domain of a single ZFN typically contains 3-6 individual zinc finger repeats, each of which can identify a sequence of, for example, 3 bp. By combining different zinc finger repeating sequences, different genome sequences can be targeted.

Meganucleases generally refer to homing endonucleases capable of identifying a nucleic acid sequence of 14-40 base in length. Long recognition sequence allows the meganucleases to have strong specificity so as to reduce the off-target effect.

âTranscription activator-like effector nucleasesâ are restriction enzymes that can be engineered to cleave specific DNA sequences, and that are typically prepared by fusing the DNA-binding domain of a transcription activator-like effector (TALE) to a DNA cleavage domain. TALE can be engineered to bind almost any desired DNA sequence.

âCRISPR systemâ generally comprises two components capable of forming complexes with sequence specificity: CRISPR nuclease or a variant thereof, and a corresponding guide RNA. Therefore, for the CRISPR system, the at least one exogenous nucleic acid sequence of interest of the invention can comprise a nucleic acid sequence encoding a CRISPR nuclease or a variant thereof, and/or a coding nucleic acid sequence of a corresponding guide RNA. Alternatively, at least one component of the gene editing system introduced into the plant cell may include a CRISPR nuclease or a variant thereof, and/or a corresponding guide RNA.

In some preferred embodiments of the present invention, the gene editing system is a CRISPR system. A large number of different CRISPR gene editing systems are known in the art, which can be applied to the invention. For example, suitable CRISPR gene editing system can be found at the Addgene company website (Addgene, Watertown, Massachusetts). CRISPR gene editing systems cover the systems capable of changing genomic sequence, but also comprise the systems for transcription control but not changing the genomic sequence.

As used herein, the term âCRISPR nucleaseâ generally refers to a nuclease present in the naturally occurring CRISPR system. The CRISPR nuclease variant comprises a modified form of natural CRISPR nuclease, artificial mutant (including the nickase mutant), catalytic active fragment, or a fusion with other functional protein/polypeptide and so on. A variety of artificial functional variants of CRISPR nuclease are known in the art, such as high specific variant or nickase variant, or a cytidine deaminase or adenosine deaminase fusion protein and so on. CRISPR nuclease or a variant thereof can interact with the corresponding guide RNA for recognizing, binding and/or cutting target nucleic acid structure. Those skilled in the art know how to select a suitable CRISPR nuclease or a variant thereof to achieve the purpose of the present invention.

CRISPR nuclease or a variant thereof used in the CRISPR gene editing system of the invention can be selected from Cas3, Cas8a, Cas5, Cas8b, Cas8c. Cas10d, Cse1, Cse2. Csy1, Csy2, Csy3; GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1 (Cas12a), C2c1, C2c3 or C2c2 protein, or functional variants of these nucleases.

In some embodiments of the present invention, the CRISPR nuclease or a variant thereof comprises a Cas9 nuclease or a variant thereof. CRISPR gene editing system based on Cas9 nuclease or variant thereof is also referred to herein as CRISPR-Cas9 gene editing system. The Cas9 nuclease can be a Cas9 nuclease from different species, such as spCas9 (having an amino acid sequence of SEQ ID NO: 15) from Streptococcus pyogenes ( S. pyogenes ).

Cas9 nuclease variant can include Cas9 nickase (nCas9), wherein one of two sub-domain (HNH nucleic acid enzyme sub-domain and RuvC sub-domain) of the Cas9 nuclease DNA cutting domain is inactivated to form a nickase. In some embodiments, combination of a Cas9 nickase and two gRNAs targeting upstream and downstream of the sequence to be edited can be used to realize deletion of the sequence to be edited, or to realize the replacement of the sequence to be edited in the presence of a donor sequence.

In some embodiments of the present invention, the CRISPR nuclease or variant thereof may also comprises Cpf1 (Cas12a) nuclease or a variant thereof such as a high specific variant. The Cpf1 nuclease can be Cpf1 nuclease from different species, such as from Francisella novicida U112; Acidaminococcus sp.BV3L6 and Lachnospiraceae bacterium ND2006. CRISPR gene editing system based on Cpf1 nuclease or variant thereof is also referred to herein as CRISPR-Cpf1 system.

In some embodiments of the present invention, the CRISPR nuclease variant further comprises a base editor. The base editor is typically a fusion protein comprising a deaminase and a CRISPR nuclease variant lack of DNA cleavage activity.

As used in the present invention, the CRISPR nuclease variant lack of DNA cleavage activity comprises but not limited to Cas9 nickase (nCas9), nuclease-dead Cas9 nuclease (dCas9) or nuclease-dead Cpf1 nuclease (dCpf1). Nuclease-dead Cas9 nuclease (dCas9) or nuclease-dead Cpf1 nuclease (dCpf1) completely lacks DNA cutting activity. A plurality of CRISPR nuclease variants lack of DNA cleavage activity are known in the art.

As used in the present invention. âdeaminaseâ refers to an enzyme that catalyzes the deamination reaction. In some embodiments of the present invention, the deaminase is s cytosine deaminase, which is capable of receiving single-stranded DNA as a substrate and capable of catalyzing cytidine or deoxycytidine respectively deaminated as uracil or deoxyuracil. In some embodiments of the present invention, the deaminase is adenosine deaminase, which is capable of receiving single-stranded DNA as a substrate and capable of catalyzing adenosine or deoxyadenosine (A) to form inosine (I). A variety of suitable cytosine deaminases or adenine deaminases with single-stranded DNA as substrate are known in the art. Suitable cytosine deaminases include, but are not limited to, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase. In some preferred embodiments, the cytosine deaminase is human APOBEC3A. Examples of suitable adenine deaminases include, but are not limited to, the DNA-dependent adenine deaminase disclosed by Nicloe M. Gaudelli et al. (doi: 10.1038/nature24644, 2017).

By using a fusion of a CRISPR nuclease variant lack of DNA cleavage activity and a deaminase (forming a so-called âbase editorâ), base editing in the target nucleotide sequence, such as conversion from C to T or conversion from A to G, can be achieved. A variety of base editors are known in the art, and those skilled in the art will know how to select a suitable base editor to achieve the object of the present invention. The base editor-based CRISPR gene editing system is also referred to as a base editing system.

In some preferred embodiments of the present invention, the CRISPR system is a base editing system. Preferably, the base editing system comprises a base editor having the amino acid sequence shown in SEQ ID NO: 14.

As used herein. âguide RNAâ and âgRNAâ can be interchangeably used, which refers to a RNA molecule that can form a complex with the CRISPR nuclease or its functional variant and is capable of targeting the complex to a target sequence because it has a certain identity to the target sequence. The guide RNA targets the target sequence through base paring between the guide RNA and the complementary strand of the target sequence. For example, gRNA used by Cas9 nuclease or its functional mutant is often composed of crRNA and tracrRNA molecules that are partially complemented to form the complex, wherein crRN A contains a guide sequence (referred to as seed sequence) that has sufficient identity to the target sequence so as to be hybridized with the complementary strand of the target sequence and directs a CRISPR complex (Cas9+crRNA+tracerRNA) to specifically bind to the target sequence. However, it has been known in the art that single guide RNA (sgRNA) can be designed, which simultaneously contains the features of crRNA and tracrRNA,

CLAIMS

Claims ( 12 )

What we claimed is:

1. A method for improving plant cell regeneration efficiency, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM, and an expression construct comprising a nucleic acid sequence of SERK, wherein the WUS comprises the amino acid sequence shown in SEQ ID NO: 1, the BBM comprises the amino acid sequence shown in SEQ ID NO: 2, or the SERK comprises the amino acid sequence shown in SEQ ID NO: 3; and/or

ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF, wherein the GRF comprises the amino acid sequence of SEQ ID NO: 7 and the GIF1 comprises the amino acid sequence shown in SEQ ID NO: 11; and/or

iii) an expression construct comprising the nucleotide sequence of SEQ ID NO: 22;

(b) regenerating an intact plant from the plant cell.

2. A method of transforming at least one exogenous nucleic acid sequence of interest into a plant, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK, wherein the WUS comprises the amino acid sequence shown in SEQ ID NO: 1, the BBM comprises the amino acid sequence shown in SEQ ID NO: 2, or the SERK comprises the amino acid sequence shown in SEQ ID NO: 3; and/or

ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF, wherein the GRF comprises the amino acid sequence of SEQ ID NO: 7, and the GIF1 comprises the amino acid sequence shown in SEQ ID NO: 11; and/or

iii) an expression construct comprising the nucleotide sequence of SEQ ID NO: 22;

(b) introducing at least one expression construct comprising the at least one exogenous nucleic acid sequence of interest into the plant cell; and

3. A method for gene editing in a plant, the method comprising:

(a) introducing into a cell of the plant

i) an expression construct comprising a coding nucleic acid sequence of WUS, an expression construct comprising a coding nucleic acid sequence of BBM and an expression construct comprising a coding nucleic acid sequence of SERK, wherein the WUS comprises the amino acid sequence shown in SEQ ID NO: 1, the BBM comprises the amino acid sequence shown in SEQ ID NO: 2, or the SERK comprises the amino acid sequence shown in SEQ ID NO: 3; and/or

ii) an expression construct comprising a coding nucleic acid sequence of GRF and an expression construct comprising a coding nucleic acid sequence of GIF, wherein the GRF comprises the amino acid sequence of SEQ ID NO: 7, and the GIF1 comprises the amino acid sequence shown in SEQ ID NO: 11; and/or

iii) an expression construct comprising the nucleotide sequence of SEQ ID NO: 22;

(b) introducing at least one expression construct comprising the at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system; or introducing at least one component of the gene editing system into the plant cell; and

4. The method according to any one of claims 1-3 , wherein the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM and the coding nucleic acid sequence of SERK are placed in a same expression construct.

5. The method according to claim 4 , wherein the coding nucleic acid sequence of WUS, the coding nucleic acid sequence of BBM, the coding nucleic acid sequence of SERK, and the at least one exogenous nucleic acid sequence of interest, if present, are placed in a same expression construct.

6. The method according to any one of claims 1-3 , wherein the coding nucleic acid sequence of GRF and the coding nucleic acid sequence of GIF are placed in a same expression construct.

7. The method according to claim 6 , wherein the coding nucleic acid sequence of GRF the coding nucleic acid sequence of GIF and the at least one exogenous nucleic acid sequence of interest, if present, are placed in a same expression construct.

8. The method according to any one of claims 1-3 , wherein the GRF comprises a mutated miRNA binding site, so as not to be regulated by the miRNA, wherein the miRNA is miR396.

9. The method according to any one of claims 1-3 , wherein the GRF is fused to the GIF by a linker, and further wherein the linker comprises an amino acid sequence comprising SEQ ID NO: 12.

10. The method according to claim 3 , wherein the gene editing system is selected from the group consisting of CRISPR system, TALEN, meganuclease and zinc finger nuclease.

11. The method according to claim 10 , wherein the gene editing system is a base editing system.

12. The method according to claim 3 , wherein the gene editing system is a base editor comprising an amino acid sequence comprising SEQ ID NO: 14.

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Inari Agriculture Technology, Inc.

Compositions and methods for soybean plant transformation

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Method for improving plant genetic transformation and gene editing efficiency — Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences (US12416013B2)

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