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CRISP_ana.pptx

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Ananya Sinha
Ananya Sinha

CRISPR/Cas systems: The link between functional genes and genetic improvement. The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding cutting-edge biotechnological tool for crop improvement Used for pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development

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CRISPR/Cas systems: The link between functional genes and genetic improvement ANANYA 1ST PhD PAMB0077
Introduction Ultimate goal of scientists and breeders: to precisely control a gene for studying its function as well as improving crop yield, quality, and tolerance to various environmental stresses The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding cutting-edge biotechnological tool for crop improvement Used for pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development
CRISPR- CAS system Clustered regularly interspaced short palindromic repeats Cas (CRISPR-associated protein) Is an adaptive phage immunity system present in archaea and bacteria.
Classification of CRISPR
CRISPR/Cas system working mechanisms and CRISPR/Cas family The commonly used Cas endonucleases, Cas9, contain two nuclease domains, RuvC and HNH, and a PAM- interacting domain (PI) RuvC and HNH domains cleave the double-strand DNA and form a double-strand break (DSB) The function of RuvC and HNH domains requires the Cas nuclease to bind a specific DNA location that are protospacer adjacent motif (PAM) dependent. After Cas cuts the DNAs, DSBs will be repaired using the cell own DNA repair mechanisms. There are two repair pathways, one is non-homologous end joining (NHEJ) repair and another one is homology-directed repair (HDR)
How does Cas nuclease precisely cut a specific sequence within the genome? One is that CRISPR/Cas system needs one gRNA that contains both crRNA and tracRNA the gRNA or sgRNA is usually a short synthetic RNA containing a scaffold tracRNA sequence and a spacer with about 20 nucleotides crRNA guides the Cas nuclease to the target sequence The tracRNA serves as scaffold for Cas enzyme binding The spacer is a sequence complementary with the target site, which is user-designed sequence and commonly called gRNA when we design the CRISPR/Cas system for gene editing
gRNA can target either strand of a gene due to the Cas enzyme have two nuclease domains, one cuts sense strand and another one cuts the anti-sense strand PAM is required for Cas enzyme function and PAM sequence serves as a binding signal for Cas nuclease; without a PAM sequence, Cas enzyme does not know where to bind and where to cut the sequence Thus, PAM sequence is required for all current CRISPR/Cas systems used for genome editing.
CRISPR/ CAS9 SYSTEM
CRISP_ana.pptx
Mechanism Double-strand-break (DSB) repair mechanisms operate in somatic cells to repair damages in nuclear DNA. DSB repair mechanisms: 1. Homologous recombination (HR): DNA templates bearing sequence similarity to the break site are used to introduce sequence changes to the target locus 2. Nonhomologous end joining (NHEJ): The broken chromosomes are rejoined, often imprecisely, thereby introducing nucleotide changes at the break site
CRISP_ana.pptx
CRISPR/Cas-based genome editing technologies serves as the bridge between functional genes and genetic improvement These technologies provide rice researchers with the opportunity to introduce specific and explicit changes at target loci and can be used for knocking out multiple genes simultaneously in the rice genome CRISPR/Cas systems: The link between functional genes and genetic Improvement- RICE AS REFERENCE
Grain yield is determined mainly by three components: panicle number, grain number per panicle, and grain weight 1.Genome editing to increase rice yield 1.1 Knockout of yield-reducing genes to increase rice yield that reduce yield in elite rice cultivars via gene-editing technology is a promising approach to the creation of ideal rice cultivars. 4 yield-reducing functional genes: GRAIN NUMBER 1a (Gn1a) DENSE AND ERECT PANICLE1 (DEP1) GRAIN SIZE 3 (GS3) IDEAL PLANT ARCHITECTURE1 (IPA1),
1.2Manipulation of regulators of cytokinin homeostasis and signal response to increase rice yield New rice germplasm with increased salinity tolerance without loss of grain yield was obtained by CRISPR/Cas9- targeted mutagenesis of the OsRR22 gene, which is involved in both cytokinin signal transduction and metabolism CRISPR-edited variants at the 30-end of OsLOGL5, which encodes a cytokinin-activation enzyme, increased grain yield under various field environments Cytokinin oxidase/dehydrogenase (CKX) is the main enzyme that inactivates cytokinin. Disruption of OsCKX11 using the CRISPR/Cas9 system increased cytokinin content and grain yield compared with wild-type rice
Strategies for improving rice yield using gene-editing technology OsCKX2/Gnla is the common target gene of two strategies (functional genes for decreasing yield and cytokinin-homeostasis and signal-response regulators)
Simultaneous mutation of rice genes encoding the abscisic acid (ABA) receptors PYRABACTIN RESISTANCE 1-LIKE 1 (PYL1), PYL4, and PYL6 yielded plants with robust growth and increased grain yield CRISPR/Cas9 induced modification of PYL9, which encodes one of the rice ABA receptors, increased drought tolerance and grain yield New rice cultivars with both high yield and high cold tolerance were obtained by simultaneously editing two yield-related negative genes (OsPIN5b and GS3) and one cold-tolerance gene (OsMYB30) using the CRISPR/Cas9 system Rice PARAQUAT TOLERANCE 3 (OsPQT3) knockout mutants generated using CRISPR/Cas9 technology conferred higher yield and increased resistance to oxidative and salt stress 1.3 Editing of plant-growth and environmental- response regulators to increase rice yield
1.4 Disruption of microRNA regulators to increase rice yield Knockout of MIR396e and MIR396f via CRISPR/Cas9 increased both grain size and panicle branching, resulting in increased grain yield Mutation in OsGRF4 introduced by CRISPR/Cas9 perturbed the miR396-directed regulation of OsGRF4 and generated plants with larger grain size and increased grain yield similar to those reported previously Knockout of UCLACYANIN 8 (OsUCL8), the downstream target of miR408, led to increased grain yield
Rice grain quality is evaluated mainly based on four quality standards: processing quality, appearance quality, eating and cooking quality (ECQ), and nutritional quality 2. Genome editing to improve rice grain quality Improvement of rice grain appearance quality, eating and cooking quality and grain nutritional quality Knockout of GS3 and GL3.1 rapidly improved grain size GS3 and Gn1a, which controls grain number, were successfully edited in four rice cultivars. Both gs3 and gs3gn1a mutants exhibited greater grain length and 1000- grain weight than their wild-type counterparts, and the gs3gn1a double mutants had more grains per panicle than the gs3 mutants
Loss-of-function mutants generated using the CRISPR/Cas9 system to target the Wx coding region in multiple rice cultivars all showed decreased AC (Amylose Content) and produced glutinous rice Knockout of the phospholipase D gene (OsPLDa1) using the CRISPR/Cas9 system produced rice grains with reduced phytic acid content compared with their wild-type counterparts Knockout of the OsNramp5 gene using the CRISPR/Cas9 system produced promising rice cultivars with extremely low cadmium accumulation in grains, without compromising yield
Genome editing to improve rice grain quality
3. Genome editing to improve rice resistance 3.1 Herbicide resistance Introduction of point mutations into the rice ALS gene using CRISPR/Cas9-mediated homologous recombination also conferred herbicide tolerance on rice plants The 548th and 627th amino acids of the rice ALS gene were edited to yield novel rice genotypes with bispyribac-sodium resistance Other amino acid substitutions in ACCase, such as W2125C and P1927F, were also obtained by CRISPR-based saturated targeted endogenous mutagenesis editors, which conferred haloxyfop resistance in rice The T102I and P106S aminoacid mutations in EPSPS and the M268T mutation in TubA2 have been reported to confer rice resistance to glyphosate and trifluralin, respectively
3.2 Disease and insect-pest resistance Li et al., 2020 used the CRISPR/Cas9 system to edit the Xa13 promoter and obtained transgene-free bacterial-blight-resistant rice CRISPR/Cas9-directed mutagenesis in the Xa13/Os8N3/OsSWEET11 coding region can also confer resistance to Xanthomonas oryzae pv. Oryzae (Xoo) CRISPR/Cas9 editing in an ethylene responsive factor, OsERF922, which is a negative regulator of blast resistance, showed increased resistance to Magnaporthe oryzae without alteration to agronomic traits Lu et al., 2018 used CRISPR/Cas9 technology to knock out the cytochrome P450 gene CYP71A1, encoding tryptamine 5- hydroxylase, which catalyzes the conversion of tryptamine to serotonin, thereby leading to rice resistance to insect pests via suppression of serotonin biosynthesis
Genome editing to improve rice resistance
Exploitation and utilization of heterosis Shen et al., 2019 rapidly created marker-free photoperiod /thermosensitive genic male sterile (P/ TGMS) rice materials by editing the male fertility gene PTGMS2-1 in two widely compatible rice cultivars Japonica photosensitive genic male-sterile rice lines were developed by targeted editing of the Carbon Starved Anther gene in japonica rice using CRISPR/ Cas9 Thermosensitive male sterile lines were also created by mutagenesis of the TMS5 gene by the CRISPR/Cas9 system Li et al., 2019 developed disease-resistant thermosensitive male- sterile rice by simultaneous genome engineering of the TMS5, Pi21, and Xa13 genes Knockout of the SaF and SaM alleles by CRISPR/Cas9 produced hybrid-compatible lines that overcame Sa-mediated hybrid male sterility in rice
Applications of CRISPR/Cas system on gene function study

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CRISP_ana.pptx

  • 1. CRISPR/Cas systems: The link between functional genes and genetic improvement ANANYA 1ST PhD PAMB0077
  • 2. Introduction Ultimate goal of scientists and breeders: to precisely control a gene for studying its function as well as improving crop yield, quality, and tolerance to various environmental stresses The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding cutting-edge biotechnological tool for crop improvement Used for pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development
  • 3. CRISPR- CAS system Clustered regularly interspaced short palindromic repeats Cas (CRISPR-associated protein) Is an adaptive phage immunity system present in archaea and bacteria.
  • 5. CRISPR/Cas system working mechanisms and CRISPR/Cas family The commonly used Cas endonucleases, Cas9, contain two nuclease domains, RuvC and HNH, and a PAM- interacting domain (PI) RuvC and HNH domains cleave the double-strand DNA and form a double-strand break (DSB) The function of RuvC and HNH domains requires the Cas nuclease to bind a specific DNA location that are protospacer adjacent motif (PAM) dependent. After Cas cuts the DNAs, DSBs will be repaired using the cell own DNA repair mechanisms. There are two repair pathways, one is non-homologous end joining (NHEJ) repair and another one is homology-directed repair (HDR)
  • 6. How does Cas nuclease precisely cut a specific sequence within the genome? One is that CRISPR/Cas system needs one gRNA that contains both crRNA and tracRNA the gRNA or sgRNA is usually a short synthetic RNA containing a scaffold tracRNA sequence and a spacer with about 20 nucleotides crRNA guides the Cas nuclease to the target sequence The tracRNA serves as scaffold for Cas enzyme binding The spacer is a sequence complementary with the target site, which is user-designed sequence and commonly called gRNA when we design the CRISPR/Cas system for gene editing
  • 7. gRNA can target either strand of a gene due to the Cas enzyme have two nuclease domains, one cuts sense strand and another one cuts the anti-sense strand PAM is required for Cas enzyme function and PAM sequence serves as a binding signal for Cas nuclease; without a PAM sequence, Cas enzyme does not know where to bind and where to cut the sequence Thus, PAM sequence is required for all current CRISPR/Cas systems used for genome editing.
  • 10. Mechanism Double-strand-break (DSB) repair mechanisms operate in somatic cells to repair damages in nuclear DNA. DSB repair mechanisms: 1. Homologous recombination (HR): DNA templates bearing sequence similarity to the break site are used to introduce sequence changes to the target locus 2. Nonhomologous end joining (NHEJ): The broken chromosomes are rejoined, often imprecisely, thereby introducing nucleotide changes at the break site
  • 12. CRISPR/Cas-based genome editing technologies serves as the bridge between functional genes and genetic improvement These technologies provide rice researchers with the opportunity to introduce specific and explicit changes at target loci and can be used for knocking out multiple genes simultaneously in the rice genome CRISPR/Cas systems: The link between functional genes and genetic Improvement- RICE AS REFERENCE
  • 13. Grain yield is determined mainly by three components: panicle number, grain number per panicle, and grain weight 1.Genome editing to increase rice yield 1.1 Knockout of yield-reducing genes to increase rice yield that reduce yield in elite rice cultivars via gene-editing technology is a promising approach to the creation of ideal rice cultivars. 4 yield-reducing functional genes: GRAIN NUMBER 1a (Gn1a) DENSE AND ERECT PANICLE1 (DEP1) GRAIN SIZE 3 (GS3) IDEAL PLANT ARCHITECTURE1 (IPA1),
  • 14. 1.2Manipulation of regulators of cytokinin homeostasis and signal response to increase rice yield New rice germplasm with increased salinity tolerance without loss of grain yield was obtained by CRISPR/Cas9- targeted mutagenesis of the OsRR22 gene, which is involved in both cytokinin signal transduction and metabolism CRISPR-edited variants at the 30-end of OsLOGL5, which encodes a cytokinin-activation enzyme, increased grain yield under various field environments Cytokinin oxidase/dehydrogenase (CKX) is the main enzyme that inactivates cytokinin. Disruption of OsCKX11 using the CRISPR/Cas9 system increased cytokinin content and grain yield compared with wild-type rice
  • 15. Strategies for improving rice yield using gene-editing technology OsCKX2/Gnla is the common target gene of two strategies (functional genes for decreasing yield and cytokinin-homeostasis and signal-response regulators)
  • 16. Simultaneous mutation of rice genes encoding the abscisic acid (ABA) receptors PYRABACTIN RESISTANCE 1-LIKE 1 (PYL1), PYL4, and PYL6 yielded plants with robust growth and increased grain yield CRISPR/Cas9 induced modification of PYL9, which encodes one of the rice ABA receptors, increased drought tolerance and grain yield New rice cultivars with both high yield and high cold tolerance were obtained by simultaneously editing two yield-related negative genes (OsPIN5b and GS3) and one cold-tolerance gene (OsMYB30) using the CRISPR/Cas9 system Rice PARAQUAT TOLERANCE 3 (OsPQT3) knockout mutants generated using CRISPR/Cas9 technology conferred higher yield and increased resistance to oxidative and salt stress 1.3 Editing of plant-growth and environmental- response regulators to increase rice yield
  • 17. 1.4 Disruption of microRNA regulators to increase rice yield Knockout of MIR396e and MIR396f via CRISPR/Cas9 increased both grain size and panicle branching, resulting in increased grain yield Mutation in OsGRF4 introduced by CRISPR/Cas9 perturbed the miR396-directed regulation of OsGRF4 and generated plants with larger grain size and increased grain yield similar to those reported previously Knockout of UCLACYANIN 8 (OsUCL8), the downstream target of miR408, led to increased grain yield
  • 18. Rice grain quality is evaluated mainly based on four quality standards: processing quality, appearance quality, eating and cooking quality (ECQ), and nutritional quality 2. Genome editing to improve rice grain quality Improvement of rice grain appearance quality, eating and cooking quality and grain nutritional quality Knockout of GS3 and GL3.1 rapidly improved grain size GS3 and Gn1a, which controls grain number, were successfully edited in four rice cultivars. Both gs3 and gs3gn1a mutants exhibited greater grain length and 1000- grain weight than their wild-type counterparts, and the gs3gn1a double mutants had more grains per panicle than the gs3 mutants
  • 19. Loss-of-function mutants generated using the CRISPR/Cas9 system to target the Wx coding region in multiple rice cultivars all showed decreased AC (Amylose Content) and produced glutinous rice Knockout of the phospholipase D gene (OsPLDa1) using the CRISPR/Cas9 system produced rice grains with reduced phytic acid content compared with their wild-type counterparts Knockout of the OsNramp5 gene using the CRISPR/Cas9 system produced promising rice cultivars with extremely low cadmium accumulation in grains, without compromising yield
  • 20. Genome editing to improve rice grain quality
  • 21. 3. Genome editing to improve rice resistance 3.1 Herbicide resistance Introduction of point mutations into the rice ALS gene using CRISPR/Cas9-mediated homologous recombination also conferred herbicide tolerance on rice plants The 548th and 627th amino acids of the rice ALS gene were edited to yield novel rice genotypes with bispyribac-sodium resistance Other amino acid substitutions in ACCase, such as W2125C and P1927F, were also obtained by CRISPR-based saturated targeted endogenous mutagenesis editors, which conferred haloxyfop resistance in rice The T102I and P106S aminoacid mutations in EPSPS and the M268T mutation in TubA2 have been reported to confer rice resistance to glyphosate and trifluralin, respectively
  • 22. 3.2 Disease and insect-pest resistance Li et al., 2020 used the CRISPR/Cas9 system to edit the Xa13 promoter and obtained transgene-free bacterial-blight-resistant rice CRISPR/Cas9-directed mutagenesis in the Xa13/Os8N3/OsSWEET11 coding region can also confer resistance to Xanthomonas oryzae pv. Oryzae (Xoo) CRISPR/Cas9 editing in an ethylene responsive factor, OsERF922, which is a negative regulator of blast resistance, showed increased resistance to Magnaporthe oryzae without alteration to agronomic traits Lu et al., 2018 used CRISPR/Cas9 technology to knock out the cytochrome P450 gene CYP71A1, encoding tryptamine 5- hydroxylase, which catalyzes the conversion of tryptamine to serotonin, thereby leading to rice resistance to insect pests via suppression of serotonin biosynthesis
  • 23. Genome editing to improve rice resistance
  • 24. Exploitation and utilization of heterosis Shen et al., 2019 rapidly created marker-free photoperiod /thermosensitive genic male sterile (P/ TGMS) rice materials by editing the male fertility gene PTGMS2-1 in two widely compatible rice cultivars Japonica photosensitive genic male-sterile rice lines were developed by targeted editing of the Carbon Starved Anther gene in japonica rice using CRISPR/ Cas9 Thermosensitive male sterile lines were also created by mutagenesis of the TMS5 gene by the CRISPR/Cas9 system Li et al., 2019 developed disease-resistant thermosensitive male- sterile rice by simultaneous genome engineering of the TMS5, Pi21, and Xa13 genes Knockout of the SaF and SaM alleles by CRISPR/Cas9 produced hybrid-compatible lines that overcame Sa-mediated hybrid male sterility in rice
  • 25. Applications of CRISPR/Cas system on gene function study