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A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
JoVE Journal
Genetics
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JoVE Journal Genetics
A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

DOI:
10.3791/56264-v

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10:32 min

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December 06, 2017

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1MCN Junior Research Group, Munich Center for Neurosciences,Ludwig-Maximilian-Universität, BioMedical Center, 2Epigenetic Engineering, Institute of Stem Cell Research,Helmholtz Zentrum, German Research Center for Environmental Health

Chapters

  • 00:05Title
  • 00:48Digestion of DNA with MNase and Separation of DNA Fragments by PAGE
  • 03:05Isolation of DNA Fragments from PAGE Gels and Linker Generation
  • 04:26End Repair of Gel-purified Fragments and Linker Ligation
  • 05:32Size Selection and Assembly of Vector-insert Construct
  • 06:18Electroporation of TG1 Electrocompetent E. coli Cells
  • 08:38Results: a gRNA Library can be Generated from and Target DNA
  • 09:56Conclusion

Summary

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Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Tags

Keywords: CRISPRGRNA LibraryGuide RNA LibraryDNA DigestionMicrococcal NucleasePAGEDNA Fragment ExtractionCORALINA MethodDNA Source Agnostic

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