Efficient CRISPR-Cas12a Base Editing Tool in Rice

Many Cas9-derived base editors have been developed for precise base editing of C-to-T and A-to-G in plants. They are usually based on SpCas9 nickase or its engineered variants. CRISPR-Cas12a is also capable of efficient multiplexed genome editing in plants, and its T-rich PAM preference can complement the G-rich PAM preference of SpCas9. However, developing efficient Cas12a base editors has been a challenge due to the lack of efficient Cas12a nickases. Due to the application of deactivated Cas12a (dCas12a), Cas12a cytosine base editors (CBEs) and adenine base editors (ABEs) have been developed in mammals. So, if a highly efficient deaminase is linked to dCas12a through an ideal linker, can an efficient Cas12a base editor be developed in plants?

On April 14, 2023, the team of Professor Qi Yiping from the University of Maryland and his collaborators published a research paper entitled "CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice" online in Plant Communications.

In this study, the researchers developed efficient Cas12a CBEs and adenine base editors (ABEs), which can be used for multiplexed genome editing in plants. Compared to Cas9 nickase-based base editors, these T-rich PAM-targeted dCas12a base editors are less likely to generate DNA breaks and are ideal for multiplexed promoter editing, that is, fine-tuning gene expression without generating indel mutations. The key to this technology lies in the efficient CRISPR-Cas12a expression system, high-efficiency LbCas12a-d156R variant, high-activity cytidine and adenine deaminase, and optimal linkers. The optimized Cas12a-CBEs can generate highly efficient monoallelic gene editing at high-activity target sites.

Four Cas12a CBEs were evaluated by next-generation sequencing (NGS) at three target sites in stable transgenic T0 rice lines using a dual Pol ll expression system and default linker 1. The CBE-based dLbCas12a-D156R outperformed other Cas12a CBEs at all three sites. Subsequently, the researchers performed multiplexed editing of four crRNAs targeting five sites using the dual Pol II promoter and the ribozyme processing system, and compared seven different flexible linkers, including the default linker 1. The multiplexed CBE T-DNA expression vectors with different linkers were evaluated by NGS in transgenic rice calli and it was observed that all Cas12a-CBEs especially with linkers 2 and 4 could perform efficient base editing at the TTTTG08 site.

Notably, the dLbCas12a-D156R ABE developed in this study appeared to be as effective as the Cas9 ABE. These highly efficient DNA break-free Cas12a CBE and ABE constructs have been entered into Addgene, a tool with great potential for singular and multiplexed base editing in plants.

Reference:

Cheng, Y.; et al. CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice. PLANT COMMUNICATIONS (2023), doi: https://doi.org/10.1016/j.xplc.2023.100601.