Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

Hu, Xixun; Meng, Xiangbing; Li, Jiayang; Wang, Kejian; Yu, Hong

doi:10.1016/j.cj.2019.06.007

Cited by 12 publications

(7 citation statements)

References 17 publications

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“…Although LbCas12a could edit genome in many dicot and monocot plants, the biallelic editing efficacy of LbCas12a remains low. For example, in LbCas12a-transformed rice, the biallelic editing frequencies were less than 50% [29,34,47]. Therefore, it is worth testing whether ttCas12a could improve biallelic editing frequencies in rice and other plants beyond citrus, Arabidopsis, and tobacco.…”

Section: Discussionmentioning

confidence: 99%

LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants

Jia

Wang

et al. 2022

Cells

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Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus.

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Section: Discussionmentioning

confidence: 99%

LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants

Jia

Wang

et al. 2022

Cells

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“…A great promise of CRISPR‐Cas technologies is the ease of multiplexed genome editing. Multiplexed plant genome‐editing systems were developed for Cas9 (Lowder et al ., 2015; Ma et al ., 2015; Xie and Minkenberg, 2015; Xing et al ., 2014), Cas12a (Hu et al ., 2019; Wang et al ., 2017, 2018; Zhang et al ., 2021b) and Cas12b (Ming et al ., 2020). In most of the cases, the goals were for simultaneous knockout of many protein‐coding genes.…”

Section: Discussionmentioning

confidence: 99%

CRISPR‐BETS: a base‐editing design tool for generating stop codons

Sretenovic

et al. 2021

Plant Biotechnology Journal

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Summary Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user‐friendly design tool named CRISPR‐BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein‐coding genes of interest. We demonstrated in rice and tomato that CRISPR‐BETS is easy‐to‐use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR‐BETS can also serve non‐plant species.

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“…These negative effects may partially explain why the multigene editing efficiency generally decreases with the number of target genes . For the CRISPR/Cas system, the disruption rates of double genes were approximately 40% in rice, compared with that of triple genes, which fell to approximately 20% . The multigene deletion in Saccharomyces cerevisiae also showed similar trends. , In addition to tRNA Gly , the other endogenous tRNAs have been used to construct the tRNA–gRNA cassettes .…”

Section: Introductionmentioning

confidence: 94%

Visualized Multigene Editing System for Aspergillus niger

Zhou

Rao

et al. 2021

ACS Synth. Biol.

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The resistance markers could ensure the entry of the CRISPR/Cas9 system into Aspergillus niger cells instead of gene editing. To increase the efficiency of positive colony screening on the primary transformation plates, we designed a visualized multigene editing system (VMS) via a unique tRNA-guide RNA (gRNA) array containing the gRNAs of a pigment gene albA and target genes. Disruption of albA produces white colonies, and the sequences of the endogenous tRNAAla, tRNAPhe, tRNAArg, tRNAIle, and tRNALeu enhance gRNA release. The disruption efficiencies of multigene were analyzed in the A. niger strain AG11 using ammA, amyA, prtT, kusA, and glaA as reporters. In white colonies on the primary transformation plates, the disruption rates of one-, two-, three-, four-, and five-target genes reached 89.2, 70.91, 50, 22.41, and 4.17%, respectively. The VMS developed here provides an effective method for screening homokaryotic multigene editing strains of A. niger.

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Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

Cited by 12 publications

References 17 publications

LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants

LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants

CRISPR‐BETS: a base‐editing design tool for generating stop codons

Visualized Multigene Editing System for Aspergillus niger

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