Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

Abstract

Summary CRISPR ‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit ( STU )‐Cas9 system, STU ‐Cas9‐ RZ , which is based on hammerhead ribozyme for processing single guide RNA s (sg RNA s). In this study, we developed two new STU ‐Cas9 systems and one STU ‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU ‐Cas9 2.0 systems process the sg RNA s with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU ‐Cas9‐Csy4 and STU ‐Cas9‐ tRNA systems showed more robust genome editing efficiencies than our first‐generation STU ‐Cas9‐ RZ system and the conventional mixed dual promoter system. We further applied the STU ‐Cas9‐ tRNA system to compare two C to T base editing systems based on rAPOBEC 1 and Pm CDA 1 cytidine deaminases. The results suggest STU ‐based Pm CDA 1 base editor system is highly efficient in rice. The STU ‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (cr RNA ) array, was also developed and demonstrated for expression of a single cr RNA and four cr RNA s. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

CRISPR‐LbCas12a‐mediated modification of citrus

“…It is worth testing whether heat shocks can enhance the mutations in LbCas12a‐transformed citrus. In addition, several novel strategies, including the ribozyme processing strategy (Tang et al ., ), the crRNA processing strategy (Wang et al ., ) and the single transcript unit strategy (Tang et al ., ; Xu et al ., ), have been developed to enhance plant genome editing. Undoubtedly, these strategies might help to optimize the efficacy of citrus genome editing in future.…”

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 expression of multiple guide RNAs for CRISPR/Cas genome editing

“…Because the tRNA sequence contains A and B boxes acting as promoter/enhancer, it increased gRNA expression in rice by one order of magnitude (Xie et al 2015). This gRNA processing strategy was later demonstrated in various plant, animal and microbial systems with consistently high efficiencies of multiplex genome editing (Dong et al 2017;Port and Bullock 2016;Shiraki and Kawakami 2018;Tang et al 2019). In addition to the tRNA-gRNA strategy, tandem repeats of gRNA-shRNA (short hairpin RNA) transcripts were also engineered for multiplex genome editing through excision of shRNAs by endogenous ribonuclease DROSHA in mammalian cells (Yan et al 2016).…”

“…HDV gene), and may provide sequence variability, which reduces gene silencing and instability. The PTG transcript can be expressed by both Pol II and Pol III promoters, and was first reported in rice by achieving indel mutation frequencies up to 100% under a single U3 promoter (Dong et al 2017;Port and Bullock 2016;Shiraki and Kawakami 2018;Tang et al 2019;Xie et al 2015). Because the tRNA sequence contains A and B boxes acting as promoter/enhancer, it increased gRNA expression in rice by one order of magnitude (Xie et al 2015).…”

“…1B). This system was shown to allow successful multiplex genome editing in animals and plants, in which a single RNA Pol II promoter is used to express Cas nuclease, gRNAs and other gRNA processing elements (e.g., ribozyme or tRNA elements) (Ding et al 2018;Tang et al 2016Tang et al , 2019Xu et al 2018;Yoshioka et al 2015). The STU system is first studied in mammalian cells, where HH-gRNA-HDV cassette is linked with Cas9 by an internal ribosome entry site (IRES) (Yoshioka et al 2015).…”