Open AccessComprehensive optimization of a reporter assay toolbox for three distinct CRISPR‐Cas systems
Author(s) -
Chen Li,
Gao Haoyuan,
Zhou Bing,
Wang Yu
Publication year - 2021
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.13198
Subject(s) - crispr , cas9 , genome editing , nuclease , reporter gene , computational biology , dna , guide rna , genome engineering , luciferase , biology , transcription activator like effector nuclease , gene targeting , chemistry , microbiology and biotechnology , genetics , gene , transfection , gene expression
The clustered, regularly interspaced, short palindromic repeats‐associated DNA nuclease (CRISPR‐Cas) protein system allows programmable gene editing through inducing double‐strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including nonhomologous DNA end joining, homology‐directed repair and single‐strand annealing. With special target design, the reporter assays as an optimized toolbox can be used to take advantage of three distinct CRISPR‐Cas systems ( Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Francisella novicida U112 Cpf1) and two different reporters (GFP and Gaussia luciferase). We further validated the Gaussia reporter assays using a series of small molecules, including NU7441, RI‐1 and Mirin, and showcased the use of a GFP reporter assay as an effective tool for enrichment of cells with edited genome.