Studying the Mechanism of CRISPR-Cas Acquisition in S. pyogenes · 2019. 9. 13. · Studying the...
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Studying the Mechanism of CRISPR-Cas Acquisition in S. pyogenes Ted Daniel Llera 1 , Shrutee Jakhanwal 2 ,Stephanie Li 2 , and Jennifer A. Doudna 2,3,4,5,6,7 1 Los Angeles Valley College, 2 Department of Molecular and Cell Biology, University of California, Berkeley, 3 Department of Chemistry, University of California, Berkeley, 4 Innovative Genomics Institute, 5 Howard Hughes Medical Institute, 6 MBIB Division, Lawrence Berkeley National Laboratory, 7 Gladstone Institutes, San Francisco Methodology Results Abstract Introduction Conclusion Acknowledgements 2019 Transfer-to-Excellence Research Experience for Undergraduates (TTE REU) Program Fig 1. Overview of CRISPR-Cas System Fig 2. Architecture of the Cas1 and Cas2 proteins. Their structure comprises four copies of Cas1 and two copies of Cas2. Cas1 proteins ar the integrases and Cas2 is a structural protein for maintain the proper orientation for protospacer integration. • In bacteria and archaea, clustered regularly interspaced short palindromic repeat (CRISPR) provide adaptive immunity against viral infection. • Universally conserved CRISPR associated (Cas) proteins, such as Cas1 and Cas2, have the ability to acquire viral DNA into the CRISPR locus as a new spacer. • In this study, we have tried to develop a screen for studying the site, sequences and orientation of the integrated protospacers in S. pyogenes. • By developing such a screen, it would create a platform to fully understand CRISPR’s evolution and further target specificity of crRNA-effector complexes. Fig 3. Full-site integration of Type V systems:. A study designed a screen for protospacer integration in which they found that V-C Cas1 has the ability to carry out full-site integration in vitro 2 . Objective In this study, we aim to develop a similar selection screen for protospacers that efficiently undergo full-site integration in Streptococcus pyogenes. Protein Purification: Transformation, over- expression, Ni-NTA, Ion exchange chromatography, and Size exclusion chromatography Acquisition Reaction: Cas1-Cas2 purified reacted with protospacer and the products were analyzed by agarose gel electrophoresis Golden Gate cloning: Acquisition reaction, electroparation, PCR gel, Mini-prep, spot-plating Figure 4 | Purification of Cas1 and Cas2. • The purification profile of size-exclusion chromatography for the Cas1 and Cas2 proteins (Fig. 4 a & b). • The purity of the purified products was assessed by performing SDS-PAGE (Fig. 4 b & d). Figure 5 | Chloramphenicol plasmid integrated to pCRISPR. • The pCRISPR containing an Ampicillin resistance gene and the donor vector for the chloramphenicol resistance gene has been depicted in Figure 2 (a) and (b) respectively. Figure 6 | Electroporation transformation. • Colonies obtained on plates containing (a) ampicillin, (b) chloramphenicol, and (c) ampicillin + chloramphenicol Results Figure 7 | Chloramphenicol was successfully integrated in pCRISPR • We performed PCR reactions with two different sets of primers that would identify the orientation of the reaction products. References [1] Nuñez, James K., et al. Foreign DNA Capture during CRISPR–Cas Adaptive Immunity. Nature, 527, 535–538. (2015) doi:10.1038/nature15760. [2] Nuñez, James K., et al. Integrase-Mediated Spacer Acquisition during CRISPR– Cas Adaptive Immunity. Nature, 519, 193–198. (2015) doi:10.1038/nature14237. I would like to thank the TTE-REU program, Nicole and Kimmy, for making this summer an unforgettable experience. To Dr. Shrutee Jakhanwal, thank you for the lessons, guidance, and support throughout the summer. To Prof. Jennifer Doudna for inspiring me to follow my dreams. To Stephanie Li and Yvette Yao for welcoming me at UC Berkeley. To the Doudna lab, especially the iBay, for providing me an incredible and life-changing opportunity. To the rest of my co-interns and E3S thank you! Support Information This research was sponsored by the Centers for Excellence in Genomic Science of the National Institutes of Health Contact Information: Ted Daniel B. Llera Email: [email protected] Phone: (818)415-2175 • We were able to successfully develop a screen protospacer integration in Type-II A system in Streptococcus pyogenes. • It is effective to test protospacer integration with purified Cas1 and Cas2 proteins • It appears to be that using the chloramphenicol cassette with Golden Gate compatible sites is successful in testing the site, orientation, and sequence of protospacer integration. Future Work • However, the screen needs to be validated further with methods such as Sanger sequencing and restriction digestion • Next-generation sequencing can also be performed on the reaction products to study a population Results Figure 8 | Cas 1 and Cas 2 protospacer integration • Cas1 and Cas2 cause the nicking of the supercoiled plasmid which then migrates slowly on the agarose gel as compared to the supercoiled plasmid. Hence, the indicative of integration
Transcript of Studying the Mechanism of CRISPR-Cas Acquisition in S. pyogenes · 2019. 9. 13. · Studying the...
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Studying the Mechanism of CRISPR-Cas Acquisition in S. pyogenesTed Daniel Llera1, Shrutee Jakhanwal2,Stephanie Li2, and Jennifer A. Doudna2,3,4,5,6,7
1Los Angeles Valley College, 2Department of Molecular and Cell Biology, University of California, Berkeley, 3Department of Chemistry, University of California, Berkeley, 4Innovative Genomics Institute, 5Howard Hughes Medical Institute, 6MBIB Division,
Lawrence Berkeley National Laboratory, 7Gladstone Institutes, San Francisco
Methodology
Results
Abstract
Introduction
Conclusion
Acknowledgements
2019 Transfer-to-Excellence Research Experience for Undergraduates (TTE REU) Program
Fig 1. Overview of CRISPR-Cas System
Fig 2. Architecture of the Cas1 and Cas2proteins. Their structure comprises four copiesof Cas1 and two copies of Cas2. Cas1 proteins arthe integrases and Cas2 is a structural protein formaintain the proper orientation for protospacerintegration.
• In bacteria and archaea, clustered regularly interspaced short palindromic repeat (CRISPR) provide adaptive immunity against viral infection.• Universally conserved CRISPR associated (Cas) proteins, such as Cas1 and Cas2, have the ability to acquire viral DNA into the CRISPR locus as a new spacer.• In this study, we have tried to develop a screen for studying the site, sequences and orientation of the integrated protospacers in S. pyogenes.• By developing such a screen, it would create a platform to fully understand CRISPR’s evolution and further target specificity of crRNA-effector complexes.
Fig 3. Full-site integration of Type Vsystems:. A study designed a screen forprotospacer integration in which they found thatV-C Cas1 has the ability to carry out full-siteintegration in vitro2.
Objective
In this study, we aim to develop a similarselection screen for protospacers that efficientlyundergo full-site integration in Streptococcuspyogenes.
Protein Purification:Transformation, over-expression, Ni-NTA, Ion exchange chromatography, and Size exclusion chromatography
Acquisition Reaction:Cas1-Cas2 purified reacted with protospacer and the products were analyzed by agarose gel electrophoresis
Golden Gate cloning:Acquisition reaction, electroparation, PCR gel, Mini-prep, spot-plating
Figure 4 | Purification of Cas1 and Cas2.• The purification profile of size-exclusion
chromatography for the Cas1 and Cas2 proteins(Fig. 4 a & b).
• The purity of the purified products was assessedby performing SDS-PAGE (Fig. 4 b & d).
Figure 5 | Chloramphenicol plasmid integrated to pCRISPR.
• The pCRISPR containing an Ampicillin resistance gene and the donor vector for the chloramphenicol resistance gene has been depicted in Figure 2 (a) and (b) respectively.
Figure 6 | Electroporation transformation.
• Colonies obtained on plates containing (a) ampicillin, (b) chloramphenicol, and (c) ampicillin + chloramphenicol
Results
Figure 7 | Chloramphenicol was successfully integrated in pCRISPR
• We performed PCR reactions with two different sets of primers that would identify the orientation of the reaction products.
References
[1] Nuñez, James K., et al. Foreign DNA Capture during CRISPR–Cas Adaptive Immunity. Nature, 527, 535–538. (2015) doi:10.1038/nature15760.[2] Nuñez, James K., et al. Integrase-Mediated Spacer Acquisition during CRISPR–Cas Adaptive Immunity. Nature, 519, 193–198. (2015) doi:10.1038/nature14237.
I would like to thank the TTE-REU program, Nicole and Kimmy, for making this summer anunforgettable experience. To Dr. Shrutee Jakhanwal, thank you for the lessons, guidance, andsupport throughout the summer. To Prof. Jennifer Doudna for inspiring me to follow my dreams.To Stephanie Li and Yvette Yao for welcoming me at UC Berkeley. To the Doudna lab, especially theiBay, for providing me an incredible and life-changing opportunity. To the rest of my co-interns andE3S thank you!
Support Information This research was sponsored by the Centers for Excellence in Genomic Science of the National Institutes of Health
Contact Information:Ted Daniel B. LleraEmail: [email protected]: (818)415-2175
• We were able to successfully develop a screenprotospacer integration in Type-II A system inStreptococcus pyogenes.
• It is effective to test protospacer integration withpurified Cas1 and Cas2 proteins
• It appears to be that using the chloramphenicolcassette with Golden Gate compatible sites issuccessful in testing the site, orientation, andsequence of protospacer integration.
Future Work
• However, the screen needs to be validated further with methods such as Sanger sequencing and restriction digestion
• Next-generation sequencing can also be performed on the reaction products to study a population
Results
Figure 8 | Cas 1 and Cas 2 protospacerintegration
• Cas1 and Cas2 cause the nicking ofthe supercoiled plasmid which thenmigrates slowly on the agarose gel ascompared to the supercoiledplasmid. Hence, the indicative ofintegration
