Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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Genome Editing as A Versatile Tool to Improve Horticultural Crop Qualities

Horticultural Plant Journal ◽

10.1016/j.hpj.2020.11.004 ◽

2020 ◽

Vol 6 (6) ◽

pp. 372-384

Author(s):

Yating Chen ◽

Wenwen Mao ◽

Ting Liu ◽

Qianqian Feng ◽

Li Li ◽

...

Keyword(s):

Genome Editing ◽

Horticultural Crop ◽

Versatile Tool

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Transcription activator-like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing

Biotechnology and Bioengineering ◽

10.1002/bit.24890 ◽

2013 ◽

Vol 110 (7) ◽

pp. 1811-1821 ◽

Cited By ~ 139

Author(s):

Ning Sun ◽

Huimin Zhao

Keyword(s):

Genome Editing ◽

Transcription Activator ◽

Highly Efficient ◽

Versatile Tool

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CRISPR-Cas Genome editing system: a versatile tool for developing disease resistant crops

Plant Stress ◽

10.1016/j.stress.2022.100056 ◽

2022 ◽

pp. 100056

Author(s):

Ashwini Talakayala ◽

Srinivas Ankanagari ◽

Mallikarjuna Garladinne

Keyword(s):

Genome Editing ◽

System A ◽

Disease Resistant ◽

Versatile Tool

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Recent advances of Cas12a applications in bacteria

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11243-9 ◽

2021 ◽

Author(s):

Meliawati Meliawati ◽

Christoph Schilling ◽

Jochen Schmid

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Bacterial Genome ◽

Adaptive Immune System ◽

Eukaryotic Cell ◽

Promising Alternative ◽

Guide Rna ◽

Recent Advances ◽

Versatile Tool

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

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SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing

Scientific Reports ◽

10.1038/s41598-017-06216-w ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 15

Author(s):

Chen Xie ◽

Yan-Lian Chen ◽

Dong-Fang Wang ◽

Yi-Lin Wang ◽

Tian-Peng Zhang ◽

...

Keyword(s):

Genome Editing ◽

Tissue Specific ◽

Versatile Tool

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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

Frontiers in Plant Science ◽

10.3389/fpls.2020.584151 ◽

2020 ◽

Vol 11 ◽

Author(s):

Anindya Bandyopadhyay ◽

Nagesh Kancharla ◽

Vivek S. Javalkote ◽

Santanu Dasgupta ◽

Thomas P. Brutnell

Keyword(s):

Genome Editing ◽

Plant Genome ◽

Versatile Tool

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Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Dictyostelium

Cells ◽

10.3390/cells8010046 ◽

2019 ◽

Vol 8 (1) ◽

pp. 46 ◽

Cited By ~ 1

Author(s):

Tetsuya Muramoto ◽

Hoshie Iriki ◽

Jun Watanabe ◽

Takefumi Kawata

Keyword(s):

Homologous Recombination ◽

Genome Editing ◽

Recent Progress ◽

Target Genes ◽

High Fidelity ◽

Target Sequence ◽

Future Perspectives ◽

Versatile Tool ◽

Endogenous Genes ◽

Genome Modifications

In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2–3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium.

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Rapid production of SaCas9 in plant-based cell-free lysate for activity testing

10.22541/au.163566964.46656930/v1 ◽

2021 ◽

Author(s):

Andreas Schiermeyer ◽

Pedro Cerda-Bennasser ◽

Thomas Schmelter ◽

Xin Huang ◽

Paul Christou ◽

...

Keyword(s):

Genome Editing ◽

Nuclease Activity ◽

In Vitro Activity ◽

Recombinant Production ◽

Cas9 Nuclease ◽

Nicotiana Tabacum L ◽

Rapid Production ◽

Versatile Tool ◽

Established Cell

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with ten different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.

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gRNA validation for wheat genome editing with the CRISPR-Cas9 system

BMC Biotechnology ◽

10.1186/s12896-019-0565-z ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Taj Arndell ◽

Niharika Sharma ◽

Peter Langridge ◽

Ute Baumann ◽

Nathan S. Watson-Haigh ◽

...

Keyword(s):

Genome Editing ◽

Hexaploid Wheat ◽

Seed Region ◽

Polyploid Species ◽

Guide Sequence ◽

Biological Target ◽

Versatile Tool ◽

Crop Genome ◽

Herbicide Glyphosate ◽

Single Mismatch

Abstract Background The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. Results The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. Conclusions We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

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