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Method Article
Ranjith Anand
Brandeis University
Corresponding Author
Gonen Memisoglu
Brandeis University
Corresponding Author
James Haber
Brandeis University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
We describe an easy, efficient, and inexpensive way to clone gRNAs into plasmid vectors carrying Cas9. The method involves designing two 25 nt complementary sequences that can be duplexed and subsequently ligated into the BplI restriction site of the plasmid vector. Using the above method, we have been able to introduce point mutations and deletions in varying sizes by using 80-nt single-stranded DNA, PCR products or gBlocks as templates.
Keywords
Cas9, gene targeting, DNA repair, DNA recombination, DNA break repair
The authors declare no conflicting financial interests.
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Cas9 mediated gene targeting cas9 mediated gene targeting Protocol for assembling gRNA into the BplI site of bRA set of plasmids
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Posted 13 Apr, 2017
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Subject Areas
Genetics
Method Article
Ranjith Anand
Brandeis University
Corresponding Author
Gonen Memisoglu
Brandeis University
Corresponding Author
James Haber
Brandeis University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 13 Apr, 2017
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Genetics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
We describe an easy, efficient, and inexpensive way to clone gRNAs into plasmid vectors carrying Cas9. The method involves designing two 25 nt complementary sequences that can be duplexed and subsequently ligated into the BplI restriction site of the plasmid vector. Using the above method, we have been able to introduce point mutations and deletions in varying sizes by using 80-nt single-stranded DNA, PCR products or gBlocks as templates.
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