Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

Yuan-Hu Jin,Young Yang,Heungsop Shin,Ruth Didier,Choogon Lee,Hyeongseok Kim,Hyunjeong Joo,Kwangjun Lee

doi:10.1038/s41598-018-36736-y

Abstract

CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.

Highlights

The prokaryotic defense system CRISPR-Cas was discovered as an adaptive immunity against foreign DNA molecules and converted to a revolutionary genome editing tool[1,2,3]
Because the circadian clock is cell autonomous, and rhythms can be measured in cells in real-time using a luciferase reporter under control of a clock promoter, clock mechanisms can be studied in cell culture models such as mouse embryonic fibroblasts (MEFs) or human cell line U2OS25–27
We showed that a complete disruption of the circadian clock can be achieved in cell culture without clonal selection and expansion by targeting a splicing site in the essential clock gene Bmal[1] using the all-in-one adenoviral CRISPR-Cas[9] system

Summary

Introduction

The prokaryotic defense system CRISPR-Cas was discovered as an adaptive immunity against foreign DNA molecules and converted to a revolutionary genome editing tool[1,2,3]. Into the host genome, reducing off-target mutations; and AV has a substantially higher genome capacity than AAV21 We have used this capacity to co-package three CRISPR-Cas[9] components along with their promoters: Cas[9], sgRNA and a fluorescent protein to aid in sorting for transduced cells. We showed that a complete disruption of the circadian clock can be achieved in cell culture without clonal selection and expansion by targeting a splicing site in the essential clock gene Bmal[1] using the all-in-one adenoviral CRISPR-Cas[9] system. We believe interrogation of gene function can be performed in diverse cell lines in a rapid manner by using our all-in-one adenoviral CRISPR-Cas[9] system since it does not involve conventional cloning, clonal selection and expansion

Methods

Results

Conclusion