Highly Efficient A·T to G·C Base Editing by Cas9n-Guided tRNA Adenosine Deaminase in Rice

Yan, Fang; Kuang, Yongjie; Ren, Bin; Wang, Jingwen; Zhang, Dawei; Lin, Honghui; Yang, Bing; Zhou, Xueping

doi:10.1016/j.molp.2018.02.008

Cited by 179 publications

(139 citation statements)

References 11 publications

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“…The efficiency of base-editing is enhanced using a nickase instead of catalytically inactive Cas9 that nicks the unmodified strand, thus stimulating mismatch repair using the unmodified strand as template [45][46][47][48]. Just recently, in addition to cytidine deaminases, adenosine deaminases could also be successfully employed for base-editing in plants [49].…”

Section: Using Cas9 As a Nickasementioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

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Section: Using Cas9 As a Nickasementioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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“…In some cases, homology directed repair (HDR) was used for targeted gene replacement (Endo et al, 2016b;Gil-Humanes et al, 2017;Li et al, 2015;Miki et al, 2018;Schiml et al, 2014;Svitashev et al, 2015). Cas9 nickase Ran et al, 2013) and base editors (Gaudelli et al, 2017;Komor et al, 2016;Li et al, 2017;Shimatani et al, 2017) have further expanded the applications of Cas9-based plant genome editing (Hua et al, 2018;Lowder et al, 2018;Lu and Zhu, 2017;Ren et al, 2017Ren et al, , 2018Yan et al, 2018;Zong et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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

Tang

Ren

Yang

et al. 2019

Plant Biotechnology Journal

119

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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.

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“…Recently, David R. Liu's group addressed this limitation by developing the adenine base editors (ABEs) that can convert AÁT to GÁC in a programmable manner in mammalian cells (Gaudelli et al, 2017). We and others showed that the ABEs can be adapted for applications in plants (Hua et al, 2018;Yan et al, 2018). The combination of adenine and cytosine base editors now can generate all four base transition mutations.…”

Section: Introductionmentioning

confidence: 99%

Expanding the base editing scope in rice by using Cas9 variants

Hua

Tao

Zhu

2018

Plant Biotechnology Journal

175

137

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Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology-directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.

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Highly Efficient A·T to G·C Base Editing by Cas9n-Guided tRNA Adenosine Deaminase in Rice

Cited by 179 publications

References 11 publications

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

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

Expanding the base editing scope in rice by using Cas9 variants

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