ABSTRACT
Abstract
A genetically modified tobacco plant or tomato plant resistant to at least one pathogenic geminiviridae virus species is provided. The plant comprises a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence that is capable of hybridizing to a nucleotide sequence of the pathogenic virus and that directs inactivation of the pathogenic virus species or plurality of viral species by the CRISPR/Cas9 system. The heterologous nucleotide sequence can be complementary to, but not limited to an Intergenic Region (IR) of the Tomato Yellow Leaf Curl Virus (TYLCV). Further provided are methods of generating a genetically modified plant that is resistant to a virus pathogen by a heterologous CRISPR/Cas9 system and expression of a gRNA specifically targeting the virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage Application of PCT/IB2016/052918, filed on May 18, 2016, which claims benefit of Application No. 62/162,856, filed on May 18, 2015 in the United States and which applications are incorporated herein by reference. A claim of priority to all, to the extent appropriate, is made.
FIELD OF THE DISCLOSURE
The present disclosure relates to methods of inhibiting viral infections of a plant by delivering a viral-specific sgRNA to a plant cell expressing Cas9 endonuclease.
SEQUENCE LISTING
The present disclosure includes a sequence listing filed in electronic form as an ASCII.txt file entitled 4053_146USWO_SL.txt, the content of which is incorporated herein by reference in its entirety. Said ASCII copy, created on Dec. 5, 2017, is 36,137 bytes in size.
BACKGROUND
Geminiviruses threaten food security and agriculture, infecting key crop species, especially in tropical and subtropical regions (Gilbertson et al., Ann. Rev. Virol 0.2, 67-93 (2015)). Geminiviruses are characterized by their twin icosahedral capsids and small, single-stranded DNA (ssDNA) genome (approximately 2.7 kb) (Hanley-Bowdoin et al., Nat. Revs. Microbiol. 11, 777-788 (2013)). A study examining genome-wide pairwise sequence identity, genome organization, host range, and insect transmission vector recently classified the family Geminiviridae into seven genera: Begomovirus, Curtovirus, Topocuvirus, Mastrevirus, Becurtovirus, Turncurtovirus , and Eragroviru (Varsani et al., Arch. Virol. 1-11 (2014)). Begomoviruses infect dicotyledonous plants via the silverleaf whitefly ( Bemisia tabaci ) vector. The genomes of Begomoviruses are composed of one (A, monopartite) or two (A and B, bipartite) components. The A and B components of the virus share a common region with nearly identical nucleotide sequences (Fondong, V. N. Mol. Plant Pathol. 14, 635-649 (2013)).
Effective strategies for controlling geminiviruses remain expensive and inefficient due to mixed virus infections and the patho-interaction of vectors, viruses, and host plants (Gilbertson et al., Development of Integrated Pest Management (IPM) Strategies for Whitefly ( Bemisia tabaci )-Transmissible Geminiviruses. 323-356 (2011)). However, recent work showed that site-specific nucleases can directly target and cleave the viral genome. This cleavage of the viral genome leads to the generation of double strand breaks (DSBs), which are either repaired by the imprecise non-homologous end-joining repair (NHEJ) machinery or by precise homology-directed repair (HDR) (Ebina et al., Sci. Rep. 3, 2510 (2013); Ali et al., Genome Biol. 16, 1-11 (2015); Aouida et al., Curr. Genet. 60, 61-74 (2014)). The presence of unrepaired DSBs ultimately leads to degradation of the virus molecules (Zaidi et al., Trends Plant Sci. 21, 279-281 (2016)). Virus variants generated by NHEJ can replicate and move systemically only if NHEJ maintains the proper âframeâ for translation and does not compromise protein function. Several site-specific nuclease platforms have been developed with potential applications for targeted interference against viral genomes.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) is an adaptive molecular immunity system used by bacteria and Archaea to fend off invading phages and conjugative plasmids (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007)). The CRISPR/Cas9 system has been harnessed for targeted mutagenesis and genome editing of eukaryotic genomes, including plants (Liu et al., Curr. Opinion Plant Biol. 30, 70-77 (2016); Nekrasov et al., Nat. Biotech. 31, 691-693 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Ali et al., Mol. Plant 8, 1288-1291 (2015)).
The CRISPR/Cas9 machinery is composed of Cas9 (a site-specific DNA endonuclease) and a synthetic single guide RNA (sgRNA, alternatively designated gRNA). The sgRNA, which carries 20-nuclecotides of target sequence information, is used to direct the Cas9 endonuclease to its genomic target sequence, which must precede a tri-nucleotide sequence known as the protospacer-associated motif (PAM). Streptomyces pyogenes Cas9 recognizes the PAM sequence NGG and cleaves three nucleotides preceding this PAM sequence on complementary and non-complementary strands (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013)). Cas9 variants with improved specificity have recently been characterized (Zetsche et al., Cell 163, 759-771 (2015); Slaymaker et al., Science 351, 84-88 (2016); Davis et al., Nat. Chem. Biol. 11, 316-318 (2015); Kleinstiver et al., Nature 523, 481-485 (2015)).
It the portability of the CRISPR/Cas9 machinery to confer molecular immunity against eukaryotic viruses, including plant DNA viruses (Karimova et al., Sci. Rep. 5, 13734 (2015); Lin et al., Mol. Ther. Nucl. Acids (2014); Ramanan et al., Sci. Reports 5, 10833 (2015); Hu et al., Proc. Natl. Acad. Sci. U.S.A. 111, 11461-11466 (2014); Chaparro-Garcia et al., Genome Biol 16, 254 (2015)). CRISPR/Cas9 machinery can target coding and non-coding sequences of different geminiviruses (Baltes et al., Nat. Plants 1, 15145 (2015); Ji et al., Nat. Plants 1, 15144 (2015)). This targeting results in reduced viral accumulation and delayed or abolished symptoms. However, different sgRNAs have different targeting efficiencies for coding or noncoding sequences. Numerous reports describe the emergence of geminiviruses with altered pathogenicity and subsequent changes in the severity of disease symptoms in infected plants, resulting from recombination-mediated genetic changes or reassortment among different viral genomes. Targeting the viral genome opens up various possibilities, including degradation and/or repair of these genomes. Under natural field conditions, where mixed viral infections exist, targeting a single virus, generating DSBs, and initiating cellular repair could induce recombination or generation of viral variants capable of replication and survival. Since the nature of the target viruses largely determines the efficiency of interference by the CRISPR/Cas9 system, production of durable resistance requires the establishment of criteria for selecting which virus sequences to target.
It has proven difficult and expensive to control or manage the disease caused by the Tomato Yellow Leaf Curl Virus (TYLCV). Several approaches for disease resistance focus on insecticide treatments of the viral transmission vector whitefly ( Bemicia tabaci ) (Lapidot et al., (2014) Advances Virus Res. 90: 147-206). Breeding for resistance is challenging due to the linkage of genes of poor fruit quality to the resistance locus. Several attempts have been made to engineer tomato for resistance to TYLCV including the over-expression of the viral proteins CP, C4, or the IR noncoding sequences (Yang et al., (2004) Phytopathol. 94: 490-496). It has been shown that binding to the origin of replication by the replication protein (Rep) interferes with the viral replication and lead to viral resistance. Synthetic zinc finger protein has been used to block the Rep protein of the beet severe curly top virus (BSCTV) from binding to the origin of replication resulting in virus resistance (Sera T. (2005) J. Virol. 79: 2614-2619). Moreover, such technology was applied to TYLCV through the interference with the Rep protein binding to the origin of replication (Koshino-Kimura et al., (2009) Nucleic Acids Symp. Series 53: 281-282; Mori et al., (2013) Mol. Biotech. 54: 198-203). Nevertheless, effective means to control or manage the TYLCV disease has proven challenging. Therefore, developing effective technologies for viral resistance is needed to increase the yield of crop species (Galvez et al., (2014) Plant Sci. 228: 11-25)).
SUMMARY
The CRISPR/Cas9 system can be used for targeted genome modification and regulation across diverse eukaryotic species. The present disclosure demonstrates the use of imparting this system for in planta viral interference. The data show that a CRISPR/Cas9 system can be used for targeted interference and cleavage of geminivirus genomes including, but not limited to, the TYLCV genome. Targeting the TYLCV IR region showed significant reduction of TYLCV accumulation and symptoms. The CRISPR/Cas9-mediated interference is virus strain-specific and can be used to target multiple viruses. The data reveal that it possible to use the CRISPR/Cas9 system to engineer plants for resistance against DNA viruses.
One aspect of the disclosure encompasses embodiments of a genetically modified plant resistant to at least one pathogenic virus species, said plant comprising a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence that is capable of hybridizing to a nucleotide sequence of the pathogenic virus under stringent conditions, or to a complement thereof, and that directs inactivation of the pathogenic virus species or plurality of viral species by the CRISPR/Cas9 system.
In some embodiments of this aspect of the disclosure, the pathogenic virus is of the geminiviridae. In some embodiments of this aspect of the disclosure, the pathogenic virus can be of the geminiviridae genus Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus.
In some embodiments of this aspect of the disclosure, the pathogenic virus can be selected from the group consisting of: Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV).
In some embodiments of this aspect of the disclosure, the plant is a tobacco plant or a tomato plant.
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of the pathogenic virus, or to a complement thereof, and wherein the nucleotide sequence of the pathogenic virus is an Intergenic Region (IR) or an Open Reading Frame (ORF).
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of an Intergenic Region (IR) of the pathogenic virus, or to a complement thereof.
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of an Intergenic Region (IR) of the Tomato Yellow Leaf Curl Virus (TYLCV), or to a complement thereof.
In some embodiments of this aspect of the disclosure, the at least one second heterologous nucleotide sequence has the nucleic acid sequence 5â²-GGCCATCCGTATAAT AT TAC-3â² (SEQ ID NO: 75).
In some embodiments, a genetically modified plant is provided according to any one or more of the foregoing embodiments.
Another aspect of the disclosure encompasses embodiments of a method of generating a genetically modified plant resistant to a virus pathogen, the method comprising the steps of: (a) obtaining a plant susceptible to a pathogenic virus, wherein said plant is genetically modified to express a heterologous CRISPR/Cas9 system; and (b) genetically modifying said plant to include at least one heterologous nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence of the pathogenic virus, or to a complement thereof, and wherein the at least one heterologous nucleotide sequence can direct inactivation of the virus pathogen by the CRISPR/Cas9 system.
BRIEF DESCRIPTION OF THE DRAWINGS
Many aspects of the disclosure can be better understood with reference to the following drawings.
FIG. 1A schematically illustrates the genome of the TYLCV and the sequences of the IR and two ORFs that were targeted with sgRNAs, according to SEQ ID NOs: 117-119, respectively, of the CRISPR/Cas9 system to reduce the infection of a plant by TYLCV.
FIG. 1B schematically illustrates the experimental protocol for demonstrating the use of CRISPR/Cas9 system to reduce viral infections in a plant.
FIG. 1C illustrates the results of a semi-quantitative PCR to determine the TYLCV titer using primers encompassing the IR region.
FIG. 1D illustrates the results of an RCA assay to test whether targeting the IR interferes with TYLCV accumulation.
FIG. 1E illustrates the results of a dot blot assay for detecting TYLCV interference.
FIG. 1F illustrates the results of Southern blotting assays demonstrating that targeting the IR of the TYLCV led to no detectable accumulation of both ssDNA and dsDNA, compared to empty vector and TYLCV.
FIG. 2A illustrates a restriction enzyme recognition site loss assay analysis showing the appearance of an SspI-resistant DNA band of 269 bp only in sgRNA-IR samples compared to vector control and TYLCV only, indicating the targeted modification of the IR region by the CRISPR/Cas9 system.
FIG. 2B illustrates that different ORFs can be targeted for modification and used to interfere with TYLCV accumulation.
FIG. 2C illustrates an alignment of Sanger sequencing reads (SEQ ID NOs: 69-74) indicating that 28% of the clones carry targeted modifications within the IR sequence.
FIG. 2D illustrates an alignment of Sanger sequencing reads (SEQ ID NOs: 62-68) indicating that different ORFs can be targeted for modification and used to interfere with TYLCV accumulation.
FIG. 3A illustrates the sequences of TYLCV (SEQ ID NO: 75) and Beet Curly Top Virus (BCTV) strain Worland-specific (SEQ ID NO: 76) IR-sgRNAs.
FIG. 3B illustrates the results of a restriction site loss assay confirming that a TYLCV-specific IR-sgRNA and not Beet Curly Top Virus (BCTV) strain Worland-specific IR-sgRNA can target any sequence similar to TYLCV IR region and thereby interfere with genome replication.
FIG. 3C illustrates alignments of Sanger sequencing reads (SEQ ID NOs: 77-88) indicating that different ORFs can be targeted for modification and used to interfere with TYLCV-specific accumulation.
FIG. 4A illustrates that WOR-RCRII-sgRNA targeted the Worland genome but not TYLCV genome.
FIG. 4B illustrates sequences SEQ ID NOs: 89-94.
FIG. 4C illustrates sequences SEQ ID NOs: 95-100.
FIG. 5A</figr
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage Application of PCT/IB2016/052918, filed on May 18, 2016, which claims benefit of Application No. 62/162,856, filed on May 18, 2015 in the United States and which applications are incorporated herein by reference. A claim of priority to all, to the extent appropriate, is made.
FIELD OF THE DISCLOSURE
The present disclosure relates to methods of inhibiting viral infections of a plant by delivering a viral-specific sgRNA to a plant cell expressing Cas9 endonuclease.
SEQUENCE LISTING
The present disclosure includes a sequence listing filed in electronic form as an ASCII.txt file entitled 4053_146USWO_SL.txt, the content of which is incorporated herein by reference in its entirety. Said ASCII copy, created on Dec. 5, 2017, is 36,137 bytes in size.
BACKGROUND
Geminiviruses threaten food security and agriculture, infecting key crop species, especially in tropical and subtropical regions (Gilbertson et al., Ann. Rev. Virol 0.2, 67-93 (2015)). Geminiviruses are characterized by their twin icosahedral capsids and small, single-stranded DNA (ssDNA) genome (approximately 2.7 kb) (Hanley-Bowdoin et al., Nat. Revs. Microbiol. 11, 777-788 (2013)). A study examining genome-wide pairwise sequence identity, genome organization, host range, and insect transmission vector recently classified the family Geminiviridae into seven genera: Begomovirus, Curtovirus, Topocuvirus, Mastrevirus, Becurtovirus, Turncurtovirus , and Eragroviru (Varsani et al., Arch. Virol. 1-11 (2014)). Begomoviruses infect dicotyledonous plants via the silverleaf whitefly ( Bemisia tabaci ) vector. The genomes of Begomoviruses are composed of one (A, monopartite) or two (A and B, bipartite) components. The A and B components of the virus share a common region with nearly identical nucleotide sequences (Fondong, V. N. Mol. Plant Pathol. 14, 635-649 (2013)).
Effective strategies for controlling geminiviruses remain expensive and inefficient due to mixed virus infections and the patho-interaction of vectors, viruses, and host plants (Gilbertson et al., Development of Integrated Pest Management (IPM) Strategies for Whitefly ( Bemisia tabaci )-Transmissible Geminiviruses. 323-356 (2011)). However, recent work showed that site-specific nucleases can directly target and cleave the viral genome. This cleavage of the viral genome leads to the generation of double strand breaks (DSBs), which are either repaired by the imprecise non-homologous end-joining repair (NHEJ) machinery or by precise homology-directed repair (HDR) (Ebina et al., Sci. Rep. 3, 2510 (2013); Ali et al., Genome Biol. 16, 1-11 (2015); Aouida et al., Curr. Genet. 60, 61-74 (2014)). The presence of unrepaired DSBs ultimately leads to degradation of the virus molecules (Zaidi et al., Trends Plant Sci. 21, 279-281 (2016)). Virus variants generated by NHEJ can replicate and move systemically only if NHEJ maintains the proper âframeâ for translation and does not compromise protein function. Several site-specific nuclease platforms have been developed with potential applications for targeted interference against viral genomes.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) is an adaptive molecular immunity system used by bacteria and Archaea to fend off invading phages and conjugative plasmids (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007)). The CRISPR/Cas9 system has been harnessed for targeted mutagenesis and genome editing of eukaryotic genomes, including plants (Liu et al., Curr. Opinion Plant Biol. 30, 70-77 (2016); Nekrasov et al., Nat. Biotech. 31, 691-693 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Ali et al., Mol. Plant 8, 1288-1291 (2015)).
The CRISPR/Cas9 machinery is composed of Cas9 (a site-specific DNA endonuclease) and a synthetic single guide RNA (sgRNA, alternatively designated gRNA). The sgRNA, which carries 20-nuclecotides of target sequence information, is used to direct the Cas9 endonuclease to its genomic target sequence, which must precede a tri-nucleotide sequence known as the protospacer-associated motif (PAM). Streptomyces pyogenes Cas9 recognizes the PAM sequence NGG and cleaves three nucleotides preceding this PAM sequence on complementary and non-complementary strands (Wright et al., Cell 164, 29-44 (2016); Hsu et al., Cell 157, 1262-1278 (2014); Barrangou et al., Science 315, 1709-1712 (2007); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013)). Cas9 variants with improved specificity have recently been characterized (Zetsche et al., Cell 163, 759-771 (2015); Slaymaker et al., Science 351, 84-88 (2016); Davis et al., Nat. Chem. Biol. 11, 316-318 (2015); Kleinstiver et al., Nature 523, 481-485 (2015)).
It the portability of the CRISPR/Cas9 machinery to confer molecular immunity against eukaryotic viruses, including plant DNA viruses (Karimova et al., Sci. Rep. 5, 13734 (2015); Lin et al., Mol. Ther. Nucl. Acids (2014); Ramanan et al., Sci. Reports 5, 10833 (2015); Hu et al., Proc. Natl. Acad. Sci. U.S.A. 111, 11461-11466 (2014); Chaparro-Garcia et al., Genome Biol 16, 254 (2015)). CRISPR/Cas9 machinery can target coding and non-coding sequences of different geminiviruses (Baltes et al., Nat. Plants 1, 15145 (2015); Ji et al., Nat. Plants 1, 15144 (2015)). This targeting results in reduced viral accumulation and delayed or abolished symptoms. However, different sgRNAs have different targeting efficiencies for coding or noncoding sequences. Numerous reports describe the emergence of geminiviruses with altered pathogenicity and subsequent changes in the severity of disease symptoms in infected plants, resulting from recombination-mediated genetic changes or reassortment among different viral genomes. Targeting the viral genome opens up various possibilities, including degradation and/or repair of these genomes. Under natural field conditions, where mixed viral infections exist, targeting a single virus, generating DSBs, and initiating cellular repair could induce recombination or generation of viral variants capable of replication and survival. Since the nature of the target viruses largely determines the efficiency of interference by the CRISPR/Cas9 system, production of durable resistance requires the establishment of criteria for selecting which virus sequences to target.
It has proven difficult and expensive to control or manage the disease caused by the Tomato Yellow Leaf Curl Virus (TYLCV). Several approaches for disease resistance focus on insecticide treatments of the viral transmission vector whitefly ( Bemicia tabaci ) (Lapidot et al., (2014) Advances Virus Res. 90: 147-206). Breeding for resistance is challenging due to the linkage of genes of poor fruit quality to the resistance locus. Several attempts have been made to engineer tomato for resistance to TYLCV including the over-expression of the viral proteins CP, C4, or the IR noncoding sequences (Yang et al., (2004) Phytopathol. 94: 490-496). It has been shown that binding to the origin of replication by the replication protein (Rep) interferes with the viral replication and lead to viral resistance. Synthetic zinc finger protein has been used to block the Rep protein of the beet severe curly top virus (BSCTV) from binding to the origin of replication resulting in virus resistance (Sera T. (2005) J. Virol. 79: 2614-2619). Moreover, such technology was applied to TYLCV through the interference with the Rep protein binding to the origin of replication (Koshino-Kimura et al., (2009) Nucleic Acids Symp. Series 53: 281-282; Mori et al., (2013) Mol. Biotech. 54: 198-203). Nevertheless, effective means to control or manage the TYLCV disease has proven challenging. Therefore, developing effective technologies for viral resistance is needed to increase the yield of crop species (Galvez et al., (2014) Plant Sci. 228: 11-25)).
SUMMARY
The CRISPR/Cas9 system can be used for targeted genome modification and regulation across diverse eukaryotic species. The present disclosure demonstrates the use of imparting this system for in planta viral interference. The data show that a CRISPR/Cas9 system can be used for targeted interference and cleavage of geminivirus genomes including, but not limited to, the TYLCV genome. Targeting the TYLCV IR region showed significant reduction of TYLCV accumulation and symptoms. The CRISPR/Cas9-mediated interference is virus strain-specific and can be used to target multiple viruses. The data reveal that it possible to use the CRISPR/Cas9 system to engineer plants for resistance against DNA viruses.
One aspect of the disclosure encompasses embodiments of a genetically modified plant resistant to at least one pathogenic virus species, said plant comprising a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence that is capable of hybridizing to a nucleotide sequence of the pathogenic virus under stringent conditions, or to a complement thereof, and that directs inactivation of the pathogenic virus species or plurality of viral species by the CRISPR/Cas9 system.
In some embodiments of this aspect of the disclosure, the pathogenic virus is of the geminiviridae. In some embodiments of this aspect of the disclosure, the pathogenic virus can be of the geminiviridae genus Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus.
In some embodiments of this aspect of the disclosure, the pathogenic virus can be selected from the group consisting of: Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV).
In some embodiments of this aspect of the disclosure, the plant is a tobacco plant or a tomato plant.
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of the pathogenic virus, or to a complement thereof, and wherein the nucleotide sequence of the pathogenic virus is an Intergenic Region (IR) or an Open Reading Frame (ORF).
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of an Intergenic Region (IR) of the pathogenic virus, or to a complement thereof.
In some embodiments of this aspect of the disclosure, the at least one heterologous nucleotide sequence is capable of hybridizing under stringent conditions to a nucleotide sequence of an Intergenic Region (IR) of the Tomato Yellow Leaf Curl Virus (TYLCV), or to a complement thereof.
In some embodiments of this aspect of the disclosure, the at least one second heterologous nucleotide sequence has the nucleic acid sequence 5â²-GGCCATCCGTATAAT AT TAC-3â² (SEQ ID NO: 75).
In some embodiments, a genetically modified plant is provided according to any one or more of the foregoing embodiments.
Another aspect of the disclosure encompasses embodiments of a method of generating a genetically modified plant resistant to a virus pathogen, the method comprising the steps of: (a) obtaining a plant susceptible to a pathogenic virus, wherein said plant is genetically modified to express a heterologous CRISPR/Cas9 system; and (b) genetically modifying said plant to include at least one heterologous nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence of the pathogenic virus, or to a complement thereof, and wherein the at least one heterologous nucleotide sequence can direct inactivation of the virus pathogen by the CRISPR/Cas9 system.
BRIEF DESCRIPTION OF THE DRAWINGS
Many aspects of the disclosure can be better understood with reference to the following drawings.
FIG. 1A schematically illustrates the genome of the TYLCV and the sequences of the IR and two ORFs that were targeted with sgRNAs, according to SEQ ID NOs: 117-119, respectively, of the CRISPR/Cas9 system to reduce the infection of a plant by TYLCV.
FIG. 1B schematically illustrates the experimental protocol for demonstrating the use of CRISPR/Cas9 system to reduce viral infections in a plant.
FIG. 1C illustrates the results of a semi-quantitative PCR to determine the TYLCV titer using primers encompassing the IR region.
FIG. 1D illustrates the results of an RCA assay to test whether targeting the IR interferes with TYLCV accumulation.
FIG. 1E illustrates the results of a dot blot assay for detecting TYLCV interference.
FIG. 1F illustrates the results of Southern blotting assays demonstrating that targeting the IR of the TYLCV led to no detectable accumulation of both ssDNA and dsDNA, compared to empty vector and TYLCV.
FIG. 2A illustrates a restriction enzyme recognition site loss assay analysis showing the appearance of an SspI-resistant DNA band of 269 bp only in sgRNA-IR samples compared to vector control and TYLCV only, indicating the targeted modification of the IR region by the CRISPR/Cas9 system.
FIG. 2B illustrates that different ORFs can be targeted for modification and used to interfere with TYLCV accumulation.
FIG. 2C illustrates an alignment of Sanger sequencing reads (SEQ ID NOs: 69-74) indicating that 28% of the clones carry targeted modifications within the IR sequence.
FIG. 2D illustrates an alignment of Sanger sequencing reads (SEQ ID NOs: 62-68) indicating that different ORFs can be targeted for modification and used to interfere with TYLCV accumulation.
FIG. 3A illustrates the sequences of TYLCV (SEQ ID NO: 75) and Beet Curly Top Virus (BCTV) strain Worland-specific (SEQ ID NO: 76) IR-sgRNAs.
FIG. 3B illustrates the results of a restriction site loss assay confirming that a TYLCV-specific IR-sgRNA and not Beet Curly Top Virus (BCTV) strain Worland-specific IR-sgRNA can target any sequence similar to TYLCV IR region and thereby interfere with genome replication.
FIG. 3C illustrates alignments of Sanger sequencing reads (SEQ ID NOs: 77-88) indicating that different ORFs can be targeted for modification and used to interfere with TYLCV-specific accumulation.
FIG. 4A illustrates that WOR-RCRII-sgRNA targeted the Worland genome but not TYLCV genome.
FIG. 4B illustrates sequences SEQ ID NOs: 89-94.
FIG. 4C illustrates sequences SEQ ID NOs: 95-100.
FIG. 5A illustrates that NB-Cas9OE plants expressing sgRNA targeting the IR region showed significantly reduced TYLCV symptoms compared with controls.
FIG. 5B illustrates the results of a Southern blot analysis confirming the absence or reduction of TYLCV genome in samples of the IR-sgRNA infiltrated plants. The results confirmed the absence, or significant reduction of, TYLCV in the IR-sgRNA targeted plants compared with the vector control.
FIG. 6 illustrates the TYLCV 2.3 genome sequence SEQ ID NO: 101. The IR, CP and RCR-II target sequences are shown in bold underlined.
FIGS. 7A-7F illustrate CRISPR/Cas9-mediated targeting of coding and non-coding sequences of the CLCuKov genome.
FIG. 7A illustrates the genome organization of Cotton Leaf Curl Kokhran Virus (CLCuKov) and Cotton Leaf Curl Multan Betasatellite (CLCuMβ). Bidirectional and overlapping ORFs (CP, Rep, Ren, TrAP, V2, and C4) are represented by arrows, the IR is represented by a box, stem loop with nonanucleotide is represented by a small circle in the IR and SCR, and targets are represented by arrowheads and individual sequences. The selected targets, one in non-coding IR, one each in coding CP or in the Rep RCRII domain, were analyzed for CRISPR/Cas9-mediated targeting followed by NHEJ repair.
FIG. 7B illustrates NHEJ repair (indel) analysis via the T7EI assay. Arrow indicates the presence of 255 bp and 191 bp regions only in samples expressing CP-sgRNA, but not in samples with TRV empty vector or virus alone.
FIG. 7C illustrates alignment of reads of PCR amplicons (SEQ ID NOs: 1-6) encompassing the CP region and subjected to Sanger sequencing for indel (NHEJ repair) confirmation.
FIG. 7D illustrates T7EI assay for detecting indels in the RCRII domain of the CLCuKov genome. The T7EI assay detected mutations only in RCRII PCR amplicons from plants infiltrated with TRV containing RCRII-sgRNA, but not in plants infiltrated with TRV empty vector or virus alone.
FIG. 7E illustrates alignment of reads of the PCR amplicons (SEQ ID NOs: 7-11) encompassing the RCRII motif and subjected to Sanger sequencing for NHEJ repair confirmation.
FIG. 7F illustrates NHEJ repair analysis at the IR sequence by restriction site loss assay. The CLCuKov IR (446 bp) was analyzed for the loss of the SspI recognition site through NHEJ (indels). The arrow indicates the expected SspI-resistant 446 bp DNA fragment; none of the samples produced the SspI-resistant DNA fragment, which is similar to TRV empty vector or virus alone.
All DNA fragments from FIGS. 7B, 7D, and 7F were resolved on a 2% agarose gel premixed with ethidium bromide stain. Arrows in FIGS. 7B, 7D, and 7F represent the expected DNA fragments. The indel percentage shown below each gel was calculated based on the Sanger sequence reads. In FIG. 7C (reverse strand sequence) and FIG. 7F , the wild-type (WT) sequences, shown at the top lines thereof, the target sequence is boxed; the protospacer-associated motif [PAM] is underlined, followed by the various indels formed, as indicated by numbers to the right of the sequence (â, deletion of x nucleotides; +, insertion of x nucleotides; and >, change of x nucleotides to y nucleotides).
FIGS. 8A-8F illustrate NHEJ repair of coding and non-coding sequences of the MeMV genome. Non-coding IR, coding CP, and the Rep RCRII domain of MeMV were analyzed for NHEJ repair.
FIG. 8A illustrates NHEJ repair (indel) analysis via an SspI recognition site loss assay. The MeMV IR (453 bp) was analyzed for the loss of the SspI recognition site at the CRISPR/Cas9 target locus. Unlike CLCuKov, NHEJ-repaired indels are indicated by arrows pointing to the 453-bp SspI-resistant DNA fragments.
FIG. 8B illustrates alignment of reads of PCR amplicons (SEQ ID NOs: 12-16) encompassing the IR of MeMV subjected to Sanger sequencing for NHEJ repair confirmation.
FIG. 8C illustrates T7EI assay to detect indels in the CP of the MeMV genome. The T7EI assay detected indels only in CP PCR amplicons from plants infiltrated with TRV containing the CP-sgRNA, but not in plants infiltrated with TRV empty vector or virus alone.
FIG. 8D illustrates alignment of reads of the PCR amplicons (SEQ ID NOs: 17-21) encompassing the target site and subjected to Sanger sequencing for NHEJ repair confirmation.
FIG. 8E illustrates NHEJ repair analysis at the RCRII motif of MeMV by T7EI assay. Arrow indicates the expected DNA fragments; TRV empty vector or virus alone did not show similar fragments.
FIG. 8F illustrates alignment of reads of the PCR amplicons (SEQ ID NOs: 22-26) encompassing the target site and subjected to Sanger sequencing for NHEJ repair confirmation.
All DNA fragments in FIGS. 8A, 8C, and 8E were resolved on a 2% agarose gel premixed with ethidium bromide stain. Arrows in FIGS. 8A, 8C, and 8E indicate the expected DNA fragments. The indel percentage shown below each gel was calculated based on the Sanger sequence reads. In FIGS. 8B, 8D, and 8F , the wild-type (WT) sequences, shown at the top lines thereof, the target sequence is boxed; the protospacer-associated motif [PAM] is underlined, followed by the various indels formed, as indicated by numbers to the right of the sequence (â, deletion of x nucleotides; +, insertion of x nucleotides; and >, change of x nucleotides to y nucleotides).
FIGS. 9A-9D illustrate variable efficiencies of indel formation at the IR sequences of different strains of TYLCV.
FIG. 9A illustrates the stem-loop structures (SEQ ID NOs: 27-32) of the different geminiviruses used in this study. The conserved nonanucleotide motif is flanked on each side by a short stretch of complementary sequences.
FIG. 9B illustrates restriction site loss assay for detecting NHEJ-based indels at the IR of TYLCSV. The TYLCSV IR (562 bp) was analyzed for the loss of the SspI recognition site at the targeting locus. The arrow indicates the location of the expected 562-bp SspI-resistant DNA fragment in samples with IR-sgRNA, but like TRV empty vector and virus alone, no SspI-resistant fragment was observed.
FIG. 9C illustrates SspI recognition site loss assay for detecting indels at the IR in TYLCV2.3. The variant TYLCSV-IRsgRNA (two lanes after marker) and authentic TYLCV2.3-IRsgRNA (last three lanes) were used to target the IR of TYLCV2.3. Arrows indicate the presence of the expected 269 bp SspI-resistant DNA fragments in samples with TYLCV2.3-IR-sgRNA or TYLCSV-IRsgRNA, but no SspI-resistant fragment was observed in TRV empty vector.
FIG. 9D illustrates a T7EI assay for detecting NHEJ-based indels in the CP and RCRII domain of the TYLCSV genome. T7EI assay detected high rates of indel formation both in CP and RCRII PCR amplicons from plants infiltrated with TRV containing CP or RCRII-sgRNA compared with TRV empty vector.
DNA fragments of FIGS. 9B, 9C, and 9D were resolved on a 2% agarose gel stained with ethidium bromide. Arrows represent the expected DNA fragment in T7EI-digested DNA. The indel percentage shown below each gel was calculated based on the Sanger sequence reads.
FIGS. 10A-10C illustrate IR targeting by CRISPR/Cas9 interferes with genome accumulation of both CLCuKov and TYLCSV.
DNA blot analysis assaying CLCuKov ( FIG. 10A ) and TYLCSV ( FIG. 10B ) genomic DNA accumulation in Cas9OE plants expressing CLCuKov-IRsgRNA and TYLCSV-IRsgRNA, respectively. CLCuKov and TYLCSV genomic DNA was detected with DIG-labeled probe produced against the respective IRs of CLCuKov and TYLCSV. All individual plants with IR-sgRNA that were infiltrated with CLCuKov and TYLCSV exhibited reduced accumulation of the genomes relative to plants inoculated with TRV empty vector and virus only. Arrowheads in FIGS. 10A and 10B indicate detection of the expected size of the TYLCV genome.
FIG. 10C illustrates an alignment of cloned Sanger-sequenced PCR amplicons (SEQ ID NOs: 33-36) encompassing the IR of CLCuKov. Alignment of sequence reads encompassing the IR shows only long deletions. The wild-type (WT) CLCuKov sequence is shown at the top; the various indels formed are indicated by numbers in the middle of the sequence reads.
FIGS. 11A-11E illustrate NHEJ-repaired CP sequence evades CRISPR/Cas9.
FIG. 11A illustrates evasion of repaired CP sequence of TYLCV genomes, as revealed by the T7EI assay. Sap from TYLCV-infected plants with an established CRISPR/Cas9 system against the CP region was applied to WT N. benthamiana plants. Total genomic DNA was isolated from top (young) leaves of sap-inoculated WT plants at 15 DAI. CP targets flanking PCR amplicons were subjected to T7EI. Arrows indicate the presence of the expected digested DNA fragment from samples of CP-targeted sap-infected plants compared to TRV empty vector with TYLCV.
FIG. 11B illustrates BsmBI-recognition site loss assay for detecting escapees. Initially, PCR amplicons were treated with BsmBI to enrich the CRISPR/Cas9 escapees. BsmBI-treated PCR fragments were used as template in another round of PCR with the same primers. Purified DNA from this PCR was again subjected to BsmBI digestion. Arrowheads indicate the expected BsmBI-resistant DNA fragments compared to WT PCR amplicons from TRV empty vector. DNA fragments of A and B were resolved on a 2% agarose gel stained with ethidium bromide.
FIG. 11C illustrates alignment of Sanger-sequencing reads of PCR amplicons (SEQ ID NOs: 37-42) encompassing the CP region of TYLCV for mutation at the CRISPR/Cas9 targeting site. The wild-type (WT) TYLCV sequence is shown at the top, the target sequence is boxed, the BsmBI site is indicated by a line, and the protospacer-associated motif [PAM] is indicated; the various mutations are shown in enlarged, bold at their respective sites.
FIG. 11D illustrates evasion analysis of TYLCSV genomes with repaired CP sequences via the T7EI assay. TYLCSV samples were prepared as in FIG. 11A . CP targets flanking the PCR amplicons were subjected to T7EI. Arrows indicate the presence of the expected digested DNA fragments from samples of CP-targeted sap-infected plants compared to TRV empty vector with TYLCSV.
FIG. 11E illustrates evasion analysis of genomes of CLCuKov genomes with repaired CP sequences via the T7EI assay. Wild-type scions were grafted to the stocks of CLCuKov-infected plants with an established CRISPR/Cas9 system against the CP region. Total genomic DNA was isolated from top (young) leaves of WT scions at 21 DAI. CP-target-flanking PCR amplicons were subjected to T7EI. Arrows indicate the presence of the expected digested DNA fragment from samples of CP-targeted CLCuKov compared to TRV empty vector with CLCuKov.
DETAILED DESCRIPTION
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, toxicology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms âa,â âan,â and âtheâ include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to âa supportâ includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, âcomprises,â âcomprising,â âcontainingâ and âhavingâ and the like can have the meaning ascribed to them in U.S. patent law and can mean âincludes,â âincluding,â and the like; âconsisting essentially ofâ or âconsists essentiallyâ or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.
Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
Definitions
The term âCRISPRâ and âCRISPR Genome Engineeringâ as used herein refers to the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system which is the most commonly used RNA-Guided Endonuclease technology for genome engineering. There are two components to this system: (1) a guide RNA and (2) an endonuclease, in this case the CRISPR associated (Cas) nuclease, Cas9.
Guide RNA is a combination of the targeting specificity of endogenous bacterial crRNA and the scaffolding properties of tracrRNA into a single chimeric guide RNA (sgRNA) transcript. When the sgRNA and the Cas9 are expressed in a cell, the genomic target sequence can be modified or permanently disrupted.
The sgRNA/Cas9 complex is recruited to a target sequence by the base-pairing between the sgRNA sequence and the complement to the target sequence in the genomic DNA. For successful activity of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the sgRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of the DNA causing a Double Strand Break (DSB).
A DSB can be repaired through one of two repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway. The NHEJ repair pathway typically results in inserts/deletions (InDels) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene. The HDR pathway requires the presence of a repair nucleic acid template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template carrying the desired change.
The term âCas9 nucleaseâ as used herein refers to an endonuclease having two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active as in wild-type Cas9, the Cas9 causes double strand breaks (DSBs) in the genomic DNA. In the absence of a suitable repair template, the DSB is then repaired by the Non-Homologous End Joining (NHEJ) DNA repair pathway. During NHEJ repair, InDels (insertions/deletions) can occur as a small number of nucleotides are either inserted or deleted at random at the DSB site, thereby altering the Open Reading Frame (ORF) of the target gene, which may significantly change the amino acid sequence downstream of the DSB. Additionally, InDels can also introduce a premature stop codon either by creating one at the DSB or by shifting the reading frame to create one downstream of the DSB. However, InDels induced by NHEJ are random, so the type and extent of gene disruption will need to be determined experimentally. To maximize the effect of gene disruption, target sequences preferably are chosen near the N-terminus of the coding region of the gene of interest, typically to introduce a DSB within the first or second exon of the gene.
The CRISPR system can also be used to introduce specific nucleotide modifications of the target sequence. Thus, cells can utilize a less error-prone DNA repair mechanism termed âHomology Directed Repair (HDR).â
To introduce nucleotide modifications to genomic DNA, a DNA repair template containing the desired sequence must be provided during HDR. The DNA template is typically transfected into a cell together with the sgRNA/Cas9 and must have a high degree of complementarity to the nucleotide sequences immediately upstream and downstream of the DSB. The length and binding position of each âhomology armâ is dependent on the size of the change being introduced. When designing a repair template for genome editing by HDR, the repair template must not contain the target sequence followed by the PAM sequence or the template itself will also be cut by the Cas9. Changing the sequence of the PAM in the repair template likely will prove sufficient to ensure it is not cut by Cas9. Again, the desired modification in the genomic DNA must be confirmed experimentally.
The term âOff-Target Effectsâ as used herein refer to when flexibility in the base-pairing interactions between the sgRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9. Single mismatches at the 5â² end of the sgRNA (furthest from the PAM site) can be permissive for off-target cleavage by Cas9.
The term ânickaseâ as used herein refers to modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-. With only one active nuclease domain, the Cas9 ânickaseâ will cut only one strand of the target DNA, thereby generating a single-strand break or ânickâ. A Cas9 nickase is still able to bind DNA based on sgRNA specificity, though nickases will only cut one of the DNA strands. The majority of CRISPR plasmids are derived from S. pyogenes and the RuvC domain inactivated, for example, by a D10A mutation.
A single-strand break, or nick, is normally repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a Double Strand Break (DSB), in what is often referred to as a âdouble nickâ or âdual nickaseâ CRISPR system. A double-nick induced DSB can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. For example, two different sgRNAs can bind in a particular genomic region. When the sgRNAs are co-expressed with a Cas9 nickase, single-strand nicks created in the DNA are quickly repaired by HDR using the intact compliment strand as a template and no change occurs. Nicks in close proximity (and on opposite strands) behave as a DSB. However, by situating two sgRNAs with target sequences in close proximity and on opposite strands of the genomic DNA, off-target effects of either sgRNA alone will result in nicks that will not change the genomic DNA. Only at the target location where both nicks are proximal, will the double nicked sequence be considered a DSB. The double-nickase technique is described in, for example, Ran et al., (2013) Cell 154: 1380-1389.
The Cas9 protein requires the targeting specificity of an sgRNA. Choosing an appropriate target sequence in the genomic DNA is a crucial step in designing an experiment. The target sequence is 20 nucleotides followed by the appropriate Protospacer Adjacent Motif (PAM) sequence in the genomic DNA. Target sequences (20 nucleotides+PAM) can be on either strand of the genomic DNA and can appear in multiple places in the genome. Accordingly a bioinformatic program is helpful to select target sequences and minimize off-target effects. There are a number of tools available to help choose/design target sequences as well as lists of bioinformatically determined (but not experimentally validated) unique sgRNAs for different genes in different species.
The term âProtospacer Adjacent Motif (PAM) Sequenceâ as used herein refers to a nucleic acid sequence present in the DNA target sequence but not in the sgRNA sequence itself. For Cas9 to successfully bind to DNA, the target sequence in the genomic DNA must be complementary to the sgRNA sequence and must be immediately followed by the correct protospacer adjacent motif or PAM sequence. Any DNA sequence with the correct target sequence followed by the PAM sequence will be bound by Cas9. A target sequence without the PAM following it is not sufficient for Cas9 to cut. Furthermore, the PAM sequence varies by the species of the bacteria from which the Cas9 was derived. The Type II CRISPR system derived from S. pyogenes , for example, has the PAM sequence NGG located on the immediate 3â² end of an sgRNA recognition sequence and components (sgRNA, Cas9) derived from different bacteria will not function together. The CRISPR system requires that both the sgRNA and Cas9 are expressed in the target cells, the respective promoters for Cas9 and sgRNA expression determining the species specificity of a particular system.
Whichever sequences and hybridization methods are used, one skilled in the art can readily determine suitable hybridization conditions, such as temperature and chemical conditions. Such hybridization methods are well known in the art. For example, for applications requiring high selectivity, one will typically desire to employ relatively stringent conditions for the hybridization reactions, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02M to about 0.10M NaCl at temperatures of about 50° C. to about 70° C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and are particularly suitable for detecting specific SNPs according to the present invention. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. Other variations in hybridization reaction conditions are well known in the art (see for example, Sambrook et al., Molecular Cloning; A Laboratory Manual 2d ed. (1989)).
The term âpolymerase chain reactionâ or âPCRâ as used herein refers to a thermocyclic, polymerase-mediated, DNA amplification reaction. A PCR typically includes template molecules, oligonucleotide primers complementary to each strand of the template molecules, a thermostable DNA polymerase, and deoxyribonucleotides, and involves three distinct processes that are multiply repeated to effect the amplification of the original nucleic acid. The three processes (denaturation, hybridization, and primer extension) are often performed at distinct temperatures, and in distinct temporal steps. In many embodiments, however, the hybridization and primer extension processes can be performed concurrently. The nucleotide sample to be analyzed may be PCR amplification products provided using the rapid cycling techniques described in U.S. Pat. Nos. 6,569,672; 6,569,627; 6,562,298; 6,556,940; 6,569,672; 6,569,627; 6,562,298; 6,556,940; 6,489,112; 6,482,615; 6,472,156; 6,413,766; 6,387,621; 6,300,124; 6,270,723; 6,245,514; 6,232,079; 6,228,634; 6,218,193; 6,210,882; 6,197,520; 6,174,670; 6,132,996; 6,126,899; 6,124,138; 6,074,868; 6,036,923; 5,985,651; 5,958,763; 5,942,432; 5,935,522; 5,897,842; 5,882,918; 5,840,573; 5,795,784; 5,795,547; 5,785,926; 5,783,439; 5,736,106; 5,720,923; 5,720,406; 5,675,700; 5,616,301; 5,576,218 and 5,455,175, the disclosures of which are incorporated by reference in their entireties. Other methods of amplification include, without limitation, NASBR, SDA, 3SR, TSA and rolling circle replication. It is understood that, in any method for producing a polynucleotide containing given modified nucleotides, one or several polymerases or amplification methods may be used. The selection of optimal polymerization conditions depends on the application.
The term âprimerâ as used herein refers to an oligonucleotide, the sequence of at least a portion of which is complementary to a segment of a template DNA which to be amplified or replicated. Typically primers are used in performing the polymerase chain reaction (PCR). A primer hybridizes with (or âannealsâ to) the template DNA and is used by the polymerase enzyme as the starting point for the replication/amplification process. By âcomplementaryâ it is meant that the nucleotide sequence of a primer is such that the primer can form a stable hydrogen bond complex with the template; i.e., the primer can hybridize or anneal to the template by virtue of the formation of base-pairs over a length of at least ten consecutive base pairs.
The terms âGeminiviridaeâ and âGeminivirusâ as used herein refer to a family of plant viruses. There are about described 325 species in this family, divided among 7 genera. Diseases associated with this family include: bright yellow mosaic, yellow mosaic, yellow mottle, leaf curling, stunting, streaks, and reduced yields. They have single-stranded circular DNA genomes encoding genes that diverge in both directions from a virion strand origin of replication (i.e. geminivirus genomes are ambisense). According to the Baltimore classification they are considered class II viruses. It is the largest known family of single stranded DNA viruses.
Mastrevirus transmission is via various leafhopper species (e.g. maize streak virus and other African streak viruses are transmitted by Cicadulina mbila), curtoviruses and the only known topocuvirus species, Tomato pseudo-curly top virus, are transmitted by treehopper species (e.g.Tomato pseudo-curly top virus is transmitted by the treehopper Micrutalis malleifera), and begomoviruses are transmitted by the whitefly species, Bemisia tabaci.
The genome can either be a single component between 2500-3100 nucleotides, or, in the case of some begomoviruses, two similar-sized components each between 2600 and 2800 nucleotides. Begomoviruses with two component (i.e. bipartite) genomes have these components separated into two different particles both of which must usually be transmitted together to initiate a new infection within a suitable host cell.
Geminivirus genomes encode only a few proteins; thus, they are dependent on host cell factors for replication: these include factors such as DNA polymerase, and probably repair polymerases, to amplify their genomes, as well as transcription factors. Geminiviruses replicate via a rolling circle mechanism like bacteriophages such as M13, and many plasmids. Replication occurs within the nucleus of an infected plant cell. First the single-stranded circular DNA is converted to a double-stranded circular intermediate. This step involves the use of cellular DNA repair enzymes to produce a complementary negative-sense strand, using the viral genomic or plus-sense DNA strand as a template. The next step is the rolling circle phase, where the viral strand is cleaved at a specific site situated within the origin of replication by the viral Rep protein in order to initiate replication. This process in a eukaryotic nucleus can give rise to concatemeric double-stranded forms of replicative intermediate genomes, although double-stranded unit circles can be isolated from infected plants and cells. New single-stranded DNA forms of the virus genome (plus-sense) are probably formed by interaction of the coat protein with replicating DNA intermediates, as genomes lacking a CP gene do not form ssDNA. The ssDNA is packaged into germinate particles in the nucleus. It is not clear if these particles can then leave the nucleus and be transmitted to surrounding cells as virions, or whether ssDNA associated with coat protein and a movement protein is the form of the genome that gets trafficked from cell to cell via the plasmodesmata.
Further definitions are provided in context below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
Abbreviations
CRISPR: Clustered Regularly Interspaced Short Palendromic Repeat (a region in bacterial genomes used in pathogen defense; crRNA: endogenous bacterial RNA that confers target specificity, requires tracrRNA to bind to Cas9; DSB: Double Strand Break; sgRNA (gRNA): guide RNA, a fusion of the crRNA and tracrRNA; sgRNA sequence: 20 nucleotides that precede a PAM sequence in the genomic DNA; HDR: Homology Directed Repair; InDel: Insertion/Deletion; NHEJ: Non-Homologous End-Joining; ORF: Open Reading Frame; PAM: Protospacer Adjacent Motif; IR, non-coding intervening region; CR, coding region; CP, coat protein
Description
The present disclosure encompasses embodiments of a method for genetically modifying a plant so as to confer to the plant resistance to a viral pathogen or a plurality of viral pathogens. It is contemplated that the plant will have been genetically modified to express the components of the CRISPR/Cas9 system by methods that are known in the art or as described in Example 7 of the present disclosure. The generation of such a plant is not restricted to only a tobacco or tomato plant but may be adapted to any plant species that is desired to modify to have a resistance to a viral pathogen.
The methods of the disclosure further include the step of identifying within the genome of a target virus pathogen a nucleotide sequence that may be isolated and delivered to the CRISPR/Cas9-expressing plant cells for an encounter with the infecting viral particles. Especially advantageously, the methods of the disclosure are useful against infections of plants by species of geminiviridae. It is contemplated that such virus pathogens can be, but are not limited to, member species of the genera Becurtovirus, Begomovirus , Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus , and most advantageously directed against the pathogens Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV). An especially significant pathogenic virus that can be targeted by the methods of the present disclosure is Tomato Yellow Leaf Curl Virus (TYLCV).
It is further contemplated that the nucleotide sequences derived from the targeted virus genome may be used to make transgenic, and thereby stably generate genetically modified plants that are able to counteract an infection by a virus pathogen. By this means, it is possible to provide a population of protected plants such that an agricultural production of the plant is resistant to the invasive virus pathogen. It is within the scope of the present disclosure, therefore, for the method to incorporate delivering the viral nucleotide sequence(s) as sgRNA by such means well-known in the art as a viral vector systemically delivered to a parent plant, by a mechanical means requiring damaging the outer wall of cells of a recipient plant tissue, by in vitro transformation and subsequent growth of the transformed cells to a mature plant, and the like.
The disclosure further provides embodiments of genetically modified plants that are transformed (most advantageously stably transformed) to express a heterologous endonuclease Cas9 and the single strands of nucleic acid that are complementary to a region of the target virus and which enable the endonuclease 9 to cleave the target viral genome, thereby inactivating the virus and reducing the level of the infection in the plant.
The CRISPR/Cas9 system provides molecular immunity to bacteria and archaea against invading phages and conjugative plasmids and has been adapted for targeted genome editing across diverse eukaryotic species. The methods of the present disclosure adapt the CRISPR/Cas9 system to function as molecular immunity machinery directed specifically against DNA viruses. The system according to the disclosure was functionally verified as operable against such as Tomato Yellow Leaf Curly Virus (TYLCV) infections. Thus, whi
CLAIMS
Claims ( 15 )
What is claimed:
1. A genetically modified plant resistant to at least one pathogenic geminivirus species, said plant comprising a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence as set forth in SEQ ID NO: 75, wherein the at least one heterologous nucleotide sequence directs inactivation of the pathogenic virus species or plurality of viral species by the CRISPR/Cas9 system.
2. The plant of claim 1 , wherein the at least one heterologous nucleotide sequence is inserted into a chromosome of the plant, is an extrachromosomal element, or is inserted in a vector.
3. The plant of claim 1 , wherein the pathogenic virus is of the geminiviridae genus Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus.
4. The plant of claim 3 , wherein the at least one pathogenic virus is selected from the group consisting of: Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV).
5. The plant of claim 1 , wherein the plant is a tobacco plant or a tomato plant.
6. The genetically modified plant of claim 1 , wherein the plant is a tobacco plant or a tomato plant, and said plant is resistant to at least one pathogenic geminivirus species selected from the group consisting of: Beet Curly Top Iran Virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV), and wherein the at least one heterologous nucleotide sequence is inserted into a chromosome of the plant, is an extrachromosomal element, or is inserted in a vector.
7. A method of generating a genetically modified plant resistant to a virus pathogen, the method comprising the steps of:
(a) obtaining a plant susceptible to a pathogenic virus, wherein said plant is genetically modified to express a heterologous CRISPR/Cas9 system; and
(b) genetically modifying said plant to have at least one heterologous nucleotide sequence as set forth in SEQ ID NO: 75, wherein the at least one heterologous nucleotide sequence can direct inactivation of the virus pathogen by the CRISPR/Cas9 system.
8. The method of claim 7 , wherein step (b) comprises delivering the at least one heterologous nucleotide sequence to a population of cells of the plant by transfection with viral vector, by a mechanical method, or by genetically modifying an isolated plant cell and generating the plant therefrom.
9. The method of claim 8 , further comprising the step of cultivating the genetically modified plant resistant to a virus pathogen to generate a population of said plants, wherein said progeny are reproduced sexually or asexually.
10. The method of claim 9 , wherein the pathogenic virus is of the geminiviridae genus Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus.
11. The method of claim 10 , wherein the pathogenic virus is selected from the group consisting of: Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV).
12. The method of claim 7 , wherein the plant is a tobacco plant or a tomato plant.
13. A method of generating a genetically modified plant resistant to a virus pathogen, the method comprising the steps of:
(a) obtaining a plant susceptible to a pathogenic geminivirus of the geminiviridae genus Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus or Turncurtovirus , and wherein said plant is genetically modified to express a heterologous CRISPR/Cas9 system;
(b) genetically modifying said plant to have at least one heterologous nucleotide sequence as set forth in SEQ ID NO: 75, wherein said at least one heterologous nucleotide sequence can direct cleavage of the virus pathogen by the CRISPR/Cas9 system, wherein step (b) comprises delivering the at least one heterologous nucleotide sequence to a population of cells of the plant by transfection with viral vector, by a mechanical method, or by genetically modifying an isolated plant cell and generating the plant therefrom; and
(c) cultivating the genetically modified plant resistant to a virus pathogen to generate a population of progenies of said plants, wherein said progeny are reproduced sexually or asexually.
14. The method of claim 13 , wherein the pathogenic virus is selected from the group consisting of: Beet Curly Top Iran virus, Spinach Severe Curly Top Virus, Bean Golden Mosaic Virus, Beet Curly Top Virus, Eragrostis curvula Streak Virus, Maize Streak Virus, Tomato Pseudo-Curly Top Virus, Turnip Curly Top Virus, and Tomato Yellow Leaf Curl Virus (TYLCV), and wherein the plant is a tobacco plant or a tomato plant.
15. The method of claim 14 , wherein the pathogenic virus is Tomato Yellow Leaf Curl Virus (TYLCV).
US15/575,749
2015-05-18
2016-05-18
Method of inhibiting plant virus pathogen infections by CRISPR/Cas9-mediated interference
Active
2036-07-29
US10662437B2
( en )
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