Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo.

Susu Wu,Yunbo Qiao,Yuting Zhao,Feiyu Chen,Liping Li,Min Li,Xiongjun Wang,Zhen Liu,Qianhui Qu,Jingli Zhong,Shiyu Sun,Xiaowen Xue,Junfan Guo

doi:10.3389/fcell.2022.809922

Abstract

CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively.

Highlights

Base editor (BE) systems, including cytosine base editors (CBEs) and adenine base editors (ABEs), can induce C-to-T and A-to-G substitutions efficiently in cultured cells, animals, and plants (Komor et al, 2016; Gaudelli et al, 2017; Kim et al, 2017; Zong et al, 2017)
We compared the protein sequences of Sth1aCas9 with Sth1Cas9 (1,121 amino acids named as St1Cas9) from Streptococcus thermophilus (Briner et al, 2014; Xu et al, 2015), showing that the two Cas9 proteins were highly similar, with only 34 different amino acids mainly distributed in protospacer adjacent motif (PAM)-interacting domains (PIDs) (Supplementary Figure S2A), which may lead to the difference of PAM recognition (Paez-Espino et al, 2015)
It demonstrated that the predicted structures of SgoCas9 and Sth1aCas9 were quite similar to that of Sth1Cas9; in particular, the structures of PIDs within these Cas9 proteins were remarkably different, which may reflect the difference of PAM recognition (Anders et al, 2014)

Summary

Introduction

Base editor (BE) systems, including cytosine base editors (CBEs) and adenine base editors (ABEs), can induce C-to-T and A-to-G substitutions efficiently in cultured cells, animals, and plants (Komor et al, 2016; Gaudelli et al, 2017; Kim et al, 2017; Zong et al, 2017). We develop two novel CBEs by fusing ancestral reconstructed APOBEC1 (ancAPOBEC1) (Koblan et al, 2018) with two Cas orthologs, SgoCas from Streptococcus_gordonii_str._Challis_substr._CH1 and Sth1aCas from Streptococcus_thermophilus_LMG_18311, respectively (Gasiunas et al, 2020), which are named as ancSgo-BE4 and ancSth1a-BE4, respectively. Both CBEs display smaller size, high activity, high fidelity, specific PAM, and editing window different from reported CBEs (Jeong et al, 2020), low byproducts for cytosine base editing, as well as partial superiority over SpCas9NG- and SpRY-based CBEs (Huang S. et al, 2019; Ren et al, 2021). These two CBEs broaden the repertoire and choice of base editing toolbox and expand the potential applications of base editors

Methods

Results

Conclusion