PAM-less plant genome editing using a CRISPR–SpRY toolbox

Ren, Qiao; Sretenovic, Simon; Liu, Shishi; Tang, Xu; Huang, Lan; He, Yao; Liu, Li; Guo, Yachong; Zhong, Zhiyong; Liu, Guanqing; Cheng, Yanhao; Zheng, Xuelian; Pan, Changtian; Yin, Desuo; Zhang, Yingxiao; Li, Wanfeng; Liu, Qi; Li, Chenghao; Qi, Yiping; Zhang, Yong

doi:10.1038/s41477-020-00827-4

Cited by 175 publications

(184 citation statements)

References 42 publications

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“…We further used three sgRNA expression cassettes to investigate the effects of sgRNA expression cassette on the editing efficiency of ABE8e ( Figure 3 a). The expression cassette of ABE8e is a regular expression cassette used in previous studies [ 14 , 15 , 19 ]. The cassette of STU-ABE8e combined the sgRNA and Cas9 into a single transcript unit (STU) ( Figure 3 a).…”

Section: Resultsmentioning

confidence: 99%

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

Wang

Xie

et al. 2021

IJMS

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Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.

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Section: Resultsmentioning

confidence: 99%

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

Wang

Xie

et al. 2021

IJMS

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“…Among engineered Cas9 variants, the near-PAMless nucleases, SpG and especially SpRY variants, present the most relaxed PAM requirements to date. The activity of these nucleases has been well described in human cell lines 6 and more recently in plants [33][34][35][36] . However, SpG and SpRY have never been applied in animals.…”

Section: Discussionmentioning

confidence: 99%

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Vicencio¹,

Moreno-Sánchez

et al. 2021

Preprint

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The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

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“…For example, Cas9-NG, ScCas9 and iSpyMacCas9 were applied to plants, using NG, NAG, and NAAR PAMs, respectively (Hua et al 2019;Sretenovic et al 2020;Wang et al 2020c). Also, the development of SpRY with very low PAM restriction was applicable for BEs in plants (Ren et al 2021;Walton et al 2020;Xu et al 2021). BE by Cas12a or its PAM altered variants has been successfully performed in human cells (Kleinstiver et al 2019;Li et al 2018a), but has not been reported in plants to date.…”

Section: Advances Of the Base Editing Techniquementioning

confidence: 99%

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Huang

Puchta

2021

Transgenic Res

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In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

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PAM-less plant genome editing using a CRISPR–SpRY toolbox

Cited by 175 publications

References 42 publications

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

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