Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

Dongfa Lin; Syeda Sadia Najam; Yu Liu; Nicola Murgia; Ilya A Vinnikov

doi:10.1016/j.mex.2023.102481

Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

MethodsX. 2023 Nov 15:12:102481. doi: 10.1016/j.mex.2023.102481. eCollection 2024 Jun.

Authors

Dongfa Lin^{1

2}, Syeda Sadia Najam², Yu Liu², Nicola Murgia², Ilya A Vinnikov²

Affiliations

¹ Key Laboratory for Molecular Enzymology and Engineering, School of Life Sciences, Jilin University, Changchun, China.
² Laboratory of Molecular Neurobiology, Sheng Yushou Center of Cell Biology and Immunology, Department of Genetics and Developmental Biology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

Abstract

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive. •All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

Keywords: CRISPR-Cas9; Homology-directed repair; Noodles: CRISPR on-target efficiency analyzer; On-target activity; Plasmid; Split luciferase.