Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing

Abstract

CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9–93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.

Computational approaches for effective CRISPR guide RNA design and evaluation

“…In theory, the CRISPR/Cas protein scans the PAM sequence, and sgRNA recognises target loci and activates endonuclease activity to cleave specific sites. However, cleavage efficiency varies greatly among different target sites and/or cell lines [14], [21], [27], [28], [51], [78], [87], [98], [99], [103], [115], [117], [122], [125], suggesting that several features may influence the binding and cutting efficacy of the sgRNA-Cas complex. Numerous studies have revealed that gRNA sequence features (sequence composition, nucleotide position, GC content), genetic and epigenetic features (chromatin accessibility, gene expression) and energetics properties (RNA secondary structure, melting temperature, free energy) all contribute to gRNA efficacy.…”

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice

“…Primers were designed and synthesized for PCR analysis ( Supplementary Table S1). Amplified products were cloned into each target site, amplified by PCR, excised by restriction digestion with the corresponding enzymes, and positive clones were selected for Sanger sequencing [53,54]. All resistant callus material used to detect mutations was also used for off-target analysis.…”

Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice

“…CRISPR‐Cas genome editing systems are becoming standard molecular tools for reverse genetics in plants. Targeted small insertions and deletions (indels) by a single‐guide RNA are often sufficient for destroying the function of a protein‐coding gene due to the capability of introducing frameshifts and early stop codons (Ren et al ., 2019; Tang et al ., 2018; Tang et al ., 2019; Zhang et al ., 2019; Zhong et al ., 2020). However, for non‐coding RNAs such as miRNAs, long non‐coding RNAs (lncRNAs) and circRNAs, introducing small indels may not yield loss of function.…”