Correction: A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants

Zhong

et al. 2020

IJMS

Self Cite

Rice (Oryza sativa) responds to various abiotic stresses during growth. Plant-specific NAM, ATAF1/2, and CUC2 (NAC) transcription factors (TFs) play an important role in controlling numerous vital growth and developmental processes. To date, 170 NAC TFs have been reported in rice, but their roles remain largely unknown. Herein, we discovered that the TF OsNAC006 is constitutively expressed in rice, and regulated by H2O2, cold, heat, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), NaCl, and polyethylene glycol (PEG) 6000 treatments. Furthermore, knockout of OsNAC006 using the CRISPR-Cas9 system resulted in drought and heat sensitivity. RNA sequencing (RNA-seq) transcriptome analysis revealed that OsNAC006 regulates the expression of genes mainly involved in response to stimuli, oxidoreductase activity, cofactor binding, and membrane-related pathways. Our findings elucidate the important role of OsNAC006 in drought responses, and provide valuable information for genetic manipulation to enhance stress tolerance in future plant breeding programs.

Section: Subcellular Localizationmentioning

confidence: 99%

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice

Zhong

et al. 2020

IJMS

Self Cite

“…Second, simultaneous repression of multiple targets with dCas9 is limited by the timeconsuming procedure of the construction of multiple independently expressed, large single guide RNA (sgRNA) expression cassettes. In contrast, a DNase-deactivated Cpf1 (ddCpf1) harboring the E1006A mutation in the RuvC domain of FnCpf1 retains precursor crRNA processing but not DNA cleavage activity and can process a single customized CRISPR array for simplified, multiplex gene expression control (42,43,56). Additionally, whole-transcriptomic analysis revealed that ddCpf1-based gene repression showed no significant off-target effects in E. coli (42).…”

Section: Resultsmentioning

confidence: 99%

CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repression in Streptomyces

Wei

Zheng

et al. 2018

Appl Environ Microbiol

117

113

has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in, it has a number of limitations, including the toxicity of Cas9 expression in some important industrial strains and the need for complex expression constructs when targeting multiple genomic loci. To address these problems, in this study, we developed a high-efficiency CRISPR-Cpf1 system (from ) for multiplex genome editing and transcriptional repression in Using an all-in-one editing plasmid with homology-directed repair (HDR), our CRISPR-Cpf1 system precisely deletes single or double genes at efficiencies of 75 to 95% in When no templates for HDR are present, random-sized DNA deletions are achieved byCpf1-induced double-strand break (DSB) repair by a reconstituted nonhomologous end joining (NHEJ) pathway. Furthermore, a DNase-deactivated Cpf1 (ddCpf1)-based integrative CRISPRi system is developed for robust, multiplex gene repression using a single customized crRNA array. Finally, we demonstrate that Cpf1 andCas9 exhibit different suitability in tested industrial species and show thatCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain SIPI-KF, in whichCas9 does not work well. Collectively, Cpf1 is a powerful and indispensable addition to the CRISPR toolbox. Rapid, efficient genetic engineering of strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency genome editing tool is established based on the CRISPR-Cpf1 system, which is an attractive and powerful alternative to the CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, Cpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance,Cpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial strains (e.g., SIPI-KF) that cannot utilize the CRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in as well as other actinomycetes.

“…The fundamental mechanistic understanding of Type V and VI systems has enabled rapid development of biotechnology tools based on Cas12a and Cas13a (Gootenberg et al, 2017; Hur et al, 2016; Tang et al, 2017; Zetsche et al, 2017). The distinct mechanisms of Cas9 and Cas12a increases the functionality of CRISPR-Cas genome editing, while the orthogonal RNA-targeting activity of Cas13a allows for tool development in creative new directions.…”

Section: Applications Based On Type V and Vi Systemsmentioning

confidence: 99%

“…Recent studies demonstrated that DNase-dead Cas12a can also be used for gene regulation in bacteria and plants (Tang et al, 2017; Zhang et al, 2017), suggesting that a wider range of applications will also be enabled by dCas12a.…”

Section: Applications Based On Type V and Vi Systemsmentioning

confidence: 99%

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

et al. 2017

CRISPR–Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR–Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR–Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR–Cas systems. This diversity has enabled further expansion of CRISPR–Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR–Cas systems.