Genome-wide specificity of prime editors in plants

Jin, Shuai; Lin, Qiupeng; Luo, Yingfeng; Zhu, Zixu; Liu, Guanwen; Li, Yunjia; Chen, Kunling; Qiu, Jian; Gao, Caixia

doi:10.1038/s41587-021-00891-x

Cited by 85 publications

(43 citation statements)

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“…Further unbiased sequencing methods that revealed all off-target positions across the genome where a guided H840A-nuclease would nick purified DNA in vitro, in most cases, did not identify any sites where the corresponding PE2-pegRNA complex acting in cells caused mutations [66]. Even genome-wide sequencing of PE-treated plants has found no evidence for off-target genome alteration by prime editors [67].…”

Section: Crispr/prime Editing For Transversion Mutations Accurate Small Insertions and Deletionsmentioning

confidence: 98%

How to increase precision in Xenopus gene editing and base editing experiments

Smith¹

2021

Preprint

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Section: Crispr/prime Editing For Transversion Mutations Accurate Small Insertions and Deletionsmentioning

confidence: 98%

How to increase precision in Xenopus gene editing and base editing experiments

Smith¹

2021

Preprint

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“…Recently, to test the precision of prime editing in plants, off-target modifications or genomic pegRNA integrations (prime editing gRNA) were measured. Such events were reported to be negligible or nonexistent [169].…”

Section: Genetic Engineeringmentioning

confidence: 98%

Genetic Approaches to Enhance Multiple Stress Tolerance in Maize

Malenica

Dunić

Vukadinović

et al. 2021

Genes

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The multiple-stress effects on plant physiology and gene expression are being intensively studied lately, primarily in model plants such as Arabidopsis, where the effects of six stressors have simultaneously been documented. In maize, double and triple stress responses are obtaining more attention, such as simultaneous drought and heat or heavy metal exposure, or drought in combination with insect and fungal infestation. To keep up with these challenges, maize natural variation and genetic engineering are exploited. On one hand, quantitative trait loci (QTL) associated with multiple-stress tolerance are being identified by molecular breeding and genome-wide association studies (GWAS), which then could be utilized for future breeding programs of more resilient maize varieties. On the other hand, transgenic approaches in maize have already resulted in the creation of many commercial double or triple stress resistant varieties, predominantly weed-tolerant/insect-resistant and, additionally, also drought-resistant varieties. It is expected that first generation gene-editing techniques, as well as recently developed base and prime editing applications, in combination with the routine haploid induction in maize, will pave the way to pyramiding more stress tolerant alleles in elite lines/varieties on time.

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“…Despite widespread applications of CRISPR technologies via NHEJ-mediated mutagenesis in basic plant research and crop improvement ( Zhu et al, 2020 ), the underlying mechanisms for variable editing efficiency and limited precise editing through HDR remain unresolved challenges for future advances ( Chechik et al, 2020 ; Zhang et al, 2021 ). Recent CRISPR-Cas9 innovations have significantly expanded the range of genetic variants by PEs ( Anzalone et al, 2019 ; Lin et al, 2020 , Jin et al, 2021 ) and enabled targeted insertion and replacement ( Lu et al, 2020 ). However, the DNA- and transgene-based approaches using Agrobacterium- and particle bombardment-mediated plant transformation still face many restrictions ( Lin et al, 2020 ; Lu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…The emerging and flexible CRISPR RNPs may offer multiple advantages in DNA-, cloning- and transgene-free genome editing with higher efficiency and precision, minimal off targets, and reduced toxicity. Furthermore, the versatile plant protoplast platform with high transfection efficiency ( Yoo et al, 2007 ; Li et al, 2013 , 2014 , 2015 ; Marx, 2016 ) is highly suitable in supporting the systematic efforts required for developing and testing new CRISPR-Cas9 designs and elucidating relevant molecular mechanisms in broad plant species ( Woo et al, 2015 ; Jin et al, 2021 ; Zhang et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

“…When combined with a specific ssODN and dual gRNAs, Cas9 RNPs enabled the generation of a precise mutation in the AtALS gene , not previously feasible in Arabidopsis protoplasts featuring low editing efficiency with plasmid DNA despite high cell quality and transfection efficiency ( Yoo et al, 2007 ; Li et al, 2013 ). Importantly, we provided the first evidence that precise mutations free of unintended indels ( Jin et al, 2021 ) could be efficiently generated by PE-mediated editing in the GFP reporter and in the Arabidopsis genome using the preassembled nCas9-RT RNP complexes without double-strand break, donor DNA or the additional DNA nick. This versatile and efficient protoplast platform will help enable plant researchers aiming to test novel Cas9-gRNA variants or create new CRISPR designs and tools for facile genome manipulation in model or crop plants.…”

Section: Introductionmentioning

confidence: 99%

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A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

2021

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The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

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Genome-wide specificity of prime editors in plants

Cited by 85 publications

References 50 publications

How to increase precision in Xenopus gene editing and base editing experiments

How to increase precision in Xenopus gene editing and base editing experiments

Genetic Approaches to Enhance Multiple Stress Tolerance in Maize

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

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