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Cold Spring Harb Protoc. 2016 May 2;2016(5). doi: 10.1101/pdb.top086793.

Characterization of Cas9-Guide RNA Orthologs.

Author information

1
Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115;
2
Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115; Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115; Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
3
Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115; Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;

Abstract

In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9-guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein-gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9-gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions.

PMID:
27140923
DOI:
10.1101/pdb.top086793
[Indexed for MEDLINE]

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