Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check.

Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. Thus, experimentally validating gRNA activity is still the gold standard in defining the best gRNAs for gene editing experiments. One such approach for experimentally selecting gRNAs with high activity is fluorescent surrogate reporter vectors. We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.

[1]  B. Liu,et al.  STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH , 2018, Cell Research.

[2]  L. Bolund,et al.  Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system , 2016, Cellular and Molecular Life Sciences.

[3]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[4]  Kristopher T. Jensen,et al.  Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells. , 2017, Journal of biotechnology.

[5]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[6]  G. Church,et al.  CRISPR-C: circularization of genes and chromosome by CRISPR in human cells , 2018, bioRxiv.

[7]  H. Wandall,et al.  Fast and sensitive detection of indels induced by precise gene targeting , 2015, Nucleic acids research.

[8]  Yunyun Cheng,et al.  Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter , 2016, Applied Biochemistry and Biotechnology.

[9]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[10]  Chase L. Beisel,et al.  Deciphering, Communicating, and Engineering the CRISPR PAM. , 2017, Journal of molecular biology.

[11]  Kristopher T. Jensen,et al.  Chromatin accessibility and guide sequence secondary structure affect CRISPR‐Cas9 gene editing efficiency , 2017, FEBS letters.