Guide RNA mCherry RNA Cas9 RNA
Undergraduate Category: Physical and Life Sciences Degree Level: Bachelor’s Degree Abstract ID# 890
Optimizing CRISPR/Cas9 Protocol for the Generation of Transgenic Axolotl Salamanders Feldman
Abstract The Axolotl salamander is an extraordinary model animal for the study of complex tissue regeneration, and offers a wealth of genetic information on how this enigmatic process occurs. By understanding this incredible phenomenon in axolotls, we advance the future development of regenerative therapies. This study focuses on a novel technique for genome editing, the CRISPR/cas9 complex. 1 We developed a scalable and cost-effective method for creating knock-out axolotls in order to functionally test candidate genes involved with regeneration.
1 O,
Monaghan
1 JR
Data
Results
Guide RNA
mCherry RNA Cas9 RNA
In-House Synthesis:
(Red Fluorescent Protein)
Cloning-Free
Method3
1. Department of Biology, Northeastern University
Screening
(Endonuclease)
Genome Editing
Targeting
Embryo Injections
Technique Guide RNA Synthesis Injection of Embryos Screening of Embryos Genotyping of Potential Knockouts
Time 1 Day 1 Day 2-3 Days 3 Weeks
Table 1. Optimized Time-Line for Generation of Transgenic Axolotls
• The estimated total cost is about $150 per gene.
Background
• Sequencing data has indicated a 66% mutation rate in endogenous genes. (Figure 7).
Fig 3. Axolotl Embryo
mCherry Screening
• Proof of Concept in Axolotl Transgene, Green Fluorescent Protein (Figures 5 and 6). Fig 4. mCherry Positive Axolotl
Conclusions
Red Fluorescence OR Phenotype Differences
This work represents one of the first axolotl knockouts using any methodology.
Fig 1. Mexican Axolotl, Ambystoma Mexicanum
Thus far, the information available to us on the genetic basis of regeneration is limited to lists of genes implicated in the process. The utility of these gene candidates is limited without the proper genetic tools to functionally test their roles during the regenerative process. 2 One powerful tool to test gene function in animals is to ablate the gene in the genome, otherwise known as a genetic “knock-out”. The RNA guided nuclease CRISPR/Cas9 system, described in Figure 2, is a recently developed gene editing technology that can ablate any gene of interest. Our goal was to develop a reliable streamlined CRISPR/Cas9 knockout methodology in the axolotl animal model to be used to gain the ability to test gene function.
Optimization of the CRISPR/Cas9 methodology in the axolotl system represents a major advance in the study of regeneration and will enable the functional testing key genes involved in the regeneration process.
Fig 5. GFP Transgenic Axolotl
Fig 6. CRISPR-Mediated Mosaic ½ GFP Knockout Axolotl
References 1. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819-823 (2013). 2. Flowers, G.P., Timberlake, A.T., McLean, K.C., Monaghan, J.R. & Crews, C.M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development (Cambridge, England) 141, 2165-2171 (2014).
Genotyping
3. Gagnon, J.A. et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PloS one 9, e98186 (2014).
Cut Site
Acknowledgements Many thanks go to the members of the Monaghan lab for their assistance with animal care and handling. Fig 2. CRISPR/cas9 Genome Editing
Fig 7. Sequencing Data representing nucleotide peaks in GPNMB Mutant
This work was supported by funding received from the College of Science, the Biology Department, and the Honors Program.
Optimizing CRISPR/Cas9 Protocol for the Generation of Transgenic Axolotl Salamanders Feldman
Abstract The Axolotl salamander is an extraordinary model animal for the study of complex tissue regeneration, and offers a wealth of genetic information on how this enigmatic process occurs. By understanding this incredible phenomenon in axolotls, we advance the future development of regenerative therapies. This study focuses on a novel technique for genome editing, the CRISPR/cas9 complex. 1 We developed a scalable and cost-effective method for creating knock-out axolotls in order to functionally test candidate genes involved with regeneration.
1 O,
Monaghan
1 JR
Data
Results
Guide RNA
mCherry RNA Cas9 RNA
In-House Synthesis:
(Red Fluorescent Protein)
Cloning-Free
Method3
1. Department of Biology, Northeastern University
Screening
(Endonuclease)
Genome Editing
Targeting
Embryo Injections
Technique Guide RNA Synthesis Injection of Embryos Screening of Embryos Genotyping of Potential Knockouts
Time 1 Day 1 Day 2-3 Days 3 Weeks
Table 1. Optimized Time-Line for Generation of Transgenic Axolotls
• The estimated total cost is about $150 per gene.
Background
• Sequencing data has indicated a 66% mutation rate in endogenous genes. (Figure 7).
Fig 3. Axolotl Embryo
mCherry Screening
• Proof of Concept in Axolotl Transgene, Green Fluorescent Protein (Figures 5 and 6). Fig 4. mCherry Positive Axolotl
Conclusions
Red Fluorescence OR Phenotype Differences
This work represents one of the first axolotl knockouts using any methodology.
Fig 1. Mexican Axolotl, Ambystoma Mexicanum
Thus far, the information available to us on the genetic basis of regeneration is limited to lists of genes implicated in the process. The utility of these gene candidates is limited without the proper genetic tools to functionally test their roles during the regenerative process. 2 One powerful tool to test gene function in animals is to ablate the gene in the genome, otherwise known as a genetic “knock-out”. The RNA guided nuclease CRISPR/Cas9 system, described in Figure 2, is a recently developed gene editing technology that can ablate any gene of interest. Our goal was to develop a reliable streamlined CRISPR/Cas9 knockout methodology in the axolotl animal model to be used to gain the ability to test gene function.
Optimization of the CRISPR/Cas9 methodology in the axolotl system represents a major advance in the study of regeneration and will enable the functional testing key genes involved in the regeneration process.
Fig 5. GFP Transgenic Axolotl
Fig 6. CRISPR-Mediated Mosaic ½ GFP Knockout Axolotl
References 1. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819-823 (2013). 2. Flowers, G.P., Timberlake, A.T., McLean, K.C., Monaghan, J.R. & Crews, C.M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development (Cambridge, England) 141, 2165-2171 (2014).
Genotyping
3. Gagnon, J.A. et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PloS one 9, e98186 (2014).
Cut Site
Acknowledgements Many thanks go to the members of the Monaghan lab for their assistance with animal care and handling. Fig 2. CRISPR/cas9 Genome Editing
Fig 7. Sequencing Data representing nucleotide peaks in GPNMB Mutant
This work was supported by funding received from the College of Science, the Biology Department, and the Honors Program.
