Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins

Jie Qiao,Lixin Ma,Siyu Lin,Wenqiang Li,Yi Liu,Wenli Sun

doi:10.1038/s42003-019-0402-x

Abstract

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)[9] ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications
The CL7 tag that engineered from the E. coli Colicin E7 DNase (CE7) retains the ultrahigh-binding affinity (KD ≈ 10−14–10−17 M) with its inhibitor Immunity protein 7 (Im7)[16], which is much higher than the His-trap approach (KD ≈ 10−8–10−9 M)
The newly synthesized Cas[9] enzymes and the transcribed single-guided RNAs (sgRNAs) were spontaneously self-assembled within E. coli cells, forming matured Cas9/sgRNA complexes

Summary

Introduction

CRISPR/Cas[9] ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. We present a streamlined method for direct production of Cas[9] RNPs from Escherichia coli by co-expression of Cas[9] and the targetspecific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas[9] and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. We achieved co-expression of Cas[9] enzymes and the associated guide RNAs in E. coli to prepare self-assembling Cas[9] RNPs15 To purify these Cas[9] RNPs, we harnessed a first NiNTA affinity purification and a following gel filtration step, resulting in a yield of ~10 mg Cas[9] RNPs from 1 L LB culture medium. We establish a valid platform to produce CRISPR/Cas RNPs at low cost and short time

Methods

Results

Conclusion