CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Moreno-Mateos, Miguel A.; Fernández, Juan Pablo; Rouet, Romain; Lane, Maura; Vejnar, Charles E.; Mis, Emily K; Khokha, Mustafa K.; Doudna, Jennifer A.; Giráldez, Antonio J.

doi:10.1101/156125

Cited by 21 publications

(33 citation statements)

References 34 publications

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“…Moreno‐Mateos et al . () showed that Cas12a activity can be substantially affected by temperature and has a higher activity at 34 °C than at 28 °C. Likewise, Li et al .…”

Section: Resultsmentioning

confidence: 99%

“…The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system represents the most widely used genome editing platform for targeted genome modifications, including plants Nekrasov et al, 2013;Sander and Joung, 2014;Shan et al, 2013). For genome editing applications, a CRISPR/Cas9 system consists of two essential components: a Cas9 effector protein, which induces blunt-end double strand breaks (DSBs), and a single-guide RNA (sgRNA), which contains an approximately 20nt targeting sequence (Hsu et al, 2014;Jinek et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Lee

Zhang

Kleinstiver

et al. 2018

Plant Biotechnology Journal

187

151

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CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

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“…Moreno‐Mateos et al . () showed that Cas12a activity can be substantially affected by temperature and has a higher activity at 34 °C than at 28 °C. Likewise, Li et al .…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Lee

Zhang

Kleinstiver

et al. 2018

Plant Biotechnology Journal

187

151

View full text Add to dashboard Cite

show abstract

“…Initial screening in human cells identified the orthologues from Acidaminococcus spec. This effect was stronger for AsCas12, explaining its reduced efficiency in plants and other organisms that grow at lower temperatures [75]. For the orthologue from Francisella novicida U112 (FnCas12a) no efficient mutagenesis could be detected at the tested target locus.…”

Section: Using Cas12a (Formerly Named Cpf1) In Plantsmentioning

confidence: 97%

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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“…Clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR‐associated (CRISPR‐Cas) nucleases are a groundbreaking genome‐engineering tool that complements classical plant breeding and transgenic methods (Moreno‐Mateos et al, ). Only two published studies in poplar have applied the CRISPR/Cas9 technology.…”

Section: Poplarmentioning

confidence: 99%

Breeding progress and preparedness for mass‐scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar

et al. 2018

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Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output–input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed‐based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass‐scale deployment of PBCs.

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CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Cited by 21 publications

References 34 publications

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

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

Breeding progress and preparedness for mass‐scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar

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