In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rahul Mahadev Shelake,Dibyajyoti Pramanik,Jae-Yean Kim

doi:10.3390/ijms23031145

Rahul Mahadev Shelake, Dibyajyoti Pramanik + Show 1 more

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https://doi.org/10.3390/ijms23031145

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Abstract

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

Highlights

Publisher’s Note: MDPI stays neutralThe adoption of the bacterial immune system, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), for targeted genetic manipulation has transformed the field of genome editing (GE) research [1]
We demonstrate the use of investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) to characterize different variants of cytidine base editor (CBE) (PmCDA1, evoCDA1, APOBEC3A) and adenine base editor (ABE) (ABE8e, ABE9e) for bacteria, exhibiting that each independent base editors (BEs) has its specific editing pattern for a given target site depending on protospacer length
Direct comparison with earlier reports is not possible due to earlier mentioned parameters, these findings demonstrate the robust applicability of the IRI-CCE platform to investigate features of broad-range CBE tools

Summary

Introduction

The adoption of the bacterial immune system, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas), for targeted genetic manipulation has transformed the field of genome editing (GE) research [1]. The CRISPR-based tools allow direct or indirect programmable engineering of all three major building blocks of life, i.e., DNA, RNA, and protein [2]. Double-strand breaks (DSBs) generated by CRISPR/Cas tool are repaired by error-prone nonhomologous end-joining (NHEJ). Most bacteria lack the efficient NHEJ pathway, restricting the DSB-dependent CRISPR/Cas applications [3]. The DSB-free DNA base editors (BEs) are emerging tools to install precise mutations at target regions in bacteria [4]. In BE systems, deaminase fusion with Cas enzyme allows precise base conversion in the targeted region. The BE tools are based on the use of nickase (nCas9) or dead Cas

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