Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9

You Kyeong Jeong,Jihyeon Yu,Sangsu Bae

doi:10.1038/s41598-019-41356-1

Abstract

Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.

Highlights

To date, a few restriction enzyme-independent methods have been developed by using the CRISPR-Cas (CRISPR associated) system[2,3,4,5,6,7,8,9]
In this study, following to the previous methods, we demonstrated a precise DNA cloning method that involves cleaving a part of the backbone plasmid using two kinds of guide RNAs with CRISPR-Cas[9] and ligating new DNA fragments using Gibson assembly[16]
To cleave the Hyg-R gene from the backbone plasmid in vitro, we treated the pXY-Hyg-AAVS1 plasmid with recombinant Cas[9] protein complexed with two single-guide RNAs that target sites flanking the Hyg-R gene within 100 bp of the Hyg-R gene boundaries in the backbone plasmid[22] (Fig. 1b and Supplementary Fig. 1)

Summary

Introduction

A few restriction enzyme-independent methods have been developed by using the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR associated) system[2,3,4,5,6,7,8,9]. The backbone plasmid lacking the Hyg-R gene and the insert DNA fragments containing Puro-R gene were mixed and re-ligated via the Gibson assembly process.

Results

Conclusion