Abstract
Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T0 transgenic plants. A proof-of-concept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.
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In recent decades, the use of CRISPR-Cas9 genome tools has led to substantial advancements in crop research. These tools have been utilized to create programmable knockout mutations or single-nucleotide polymorphisms (SNPs) in the gene coding region, aiming to improve specific traits (Gürel et al. 2020). However, considering that agronomically important traits are mostly quantitative, there is growing interest in editing promoters to alter the strength of gene expression (Shi et al. 2023). Eukaryotic promoters consist of a core promoter region and upstream cis-regulatory elements (CREs). Successful editing of CREs has created a series of desirable traits that were previously absent in natural resources (Liu et al. 2021; Rodríguez-Leal et al. 2017; Song et al. 2022; Zhou et al. 2023). Nevertheless, achieving gene expression alterations through the targeting of multiple CREs is often laborious. Alternatively, minimal mutations in the core promoter region, which are replete with short TA-rich sequences, can induce significant changes in the abundance of downstream gene transcripts. Unfortunately, due to the lack of an appropriate protospacer adjacent motif (PAM), G-rich PAM recognition Cas9 enzymes, such as Streptococcus pyogenes Cas9 (SpCas9), are rarely used for core promoter editing.
Various Cas12a proteins and members of the Cas12 family preferentially utilize T-rich PAMs (Gürel et al. 2020). Although CRISPR-Cas12 systems have been extensively applied for single- and multiple-gene editing, base editing, gene activation, transcriptional repression, epigenome editing, and promoter editing (Cheng et al. 2023; Liu et al. 2022; Ming et al. 2020; Zhang et al. 2021; Zhou et al. 2023), the scope may be restricted by the requirement of relatively longer PAMs. The Cas9 gene of the probiotic Lactobacillus rhamnosus (LrCas9) and the hybrid SpCas9–Streptococcus macacae Cas9 enzyme (iSpyMacCas9) have expanded the editing scope to include NGAAA and NAA PAMs in crops (Sretenovic et al. 2021; Zhong et al. 2019). For the albino PDS-T lines and other representative edited plants, Sanger sequencing was performed. Some chromatographic overlaps were resolved through sequencing TA clones of the target amplicon. All primers used in this study are listed in Supplemental Table S4.
Gene expression analysis
The expression of WX was analyzed in the grains of the wild-type (WT) and T-DNA-free homozygous T1 mutant plants after 14 days of filling. Three independent lines of each allele were used as biological replicates. Total RNA was extracted from ~ 0.2 g of sample using TRIzol reagent (Invitrogen, Carlsbad, USA). Reverse transcription of cDNA was performed using HiScript III All-in-one RT SuperMix Perfect (Vazyme, Nan**g, China). Quantitative PCR was conducted using the qTOWER 2.2 system (Analytic Jena, Jena, Germany) and M5 HiPer Realtime PCR Super Mix (Mei5bio, Bei**g, China). Relative gene expression levels were calculated using the 2-∆∆CT algorithm, with the rice ACTIN2 gene serving as the internal control.
Determination of amylose content
The mature seeds were ground into flour using steel beads. Approximately, 0.1 g of flour from a sample was dissolved in a 0.09% sodium hydroxide solution. The amylose content was then determined through flow injection analysis with an iodine reagent using an automatic analyzer (HACH, Loveland, USA). The absorbances of the resulting colors were recorded at 720 nm for content calculation.