In Vitro Pre-validation of Gene Editing by CRISPR/Cas9 Ribonucleoprotein

BACKGROUND: The CRISPR/Cas9 genome editing system is a powerful and simple gene editing method. The format of the CRISPR components is one of the important factors in targeting efficiency. Compared to plasmid or mRNA (IVTs) format, using the CRISPR/Cas9 system as Cas9–crRNA–tracrRNA RNP format is more efficient and rapid, especially in minimizing some of the pitfalls of CRISPR-mediated gene editing. In addition to efficient in vivo applications of the CRISPR RNP format in a variety of cell types and organisms, another advantage of this approach is usability for in vitro applications in which the crRNAs in the tracrRNA–crRNA structure guides the Mg2+-dependent RNAdirected DNA endonuclease to introduce double-strand breaks at specific sites in DNA. METHODS: Here, Cas9–crRNA–tracrRNA RNP system was used to test the designed crRNAs for in vitro DNA cleavage by Cas9 protein in RAG1, RAG2 and IL2RG genes. RESULTS: The results of cleavage reveal theCas9–crRNA–tracrRNA RNP system is a rapid and efficient way to pre-validate the efficiency of CRISPR cleavage with crRNAs designed for RAG1, RAG2 and IL2RG genes. CONCLUSION: one step in vitro cleavage of DNA by CRISPR/Cas9 ribonucleoprotein complex can be used to pre-validate the functionality and relative efficiency of CRISPR system for targeting genes.