Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells

Bigelyte, Greta; Young, Joshua K.; Karvelis, Tautvydas; Budre, Karolina; Zedaveinyte, Rimante; Djukanovic, Vesna; Ginkel, Elizabeth Van; Paulraj, Sushmitha; Gasior, Stephen L.; Jones, Spencer; Feigenbutz, Lanie; Clair, Grace St.; Barone, Pierluigi; Bohn, Jennifer; Acharya, Ananta; Zastrow-Hayes, Gina; Henkel-Heinecke, Selgar; Šilanskas, Arūnas; Seidel, Ralf; Šikšnys, Virginijus

doi:10.1038/s41467-021-26469-4

Cited by 63 publications

(50 citation statements)

References 49 publications

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“…Success here is leading to a new class of therapeutic interventions ( 15–17 ) to confront diseases ‘ upstream ’ by targeting their genomic origin, with precise molecular destruction of pathogenic RNAs before they translate into the disease. Over recent decades, powerful and versatile sequence-specific interventions, such as siRNAs ( 18–24 ) CRISPR ( 25–27 ) and antisense oligonucleotides (ASOs) ( 28–41 ) have emerged for genome editing and for manipulation of gene expression, reliant upon the precision of Watson-Crick base-pairing between short RNA or DNA guide sequences and target nucleic acids. Recent advances have enhanced their serum stability, safety profiles and improved cell-targeting capabilities ( 22–26 , 31 ).…”

Section: Introductionmentioning

confidence: 99%

“Bind, cleave and leave”: multiple turnover catalysis of RNA cleavage by bulge–loop inducing supramolecular conjugates

Amirloo

Staroseletz

Yousaf

et al. 2021

Nucleic Acids Research

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Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge–loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge–loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

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Section: Introductionmentioning

confidence: 99%

“Bind, cleave and leave”: multiple turnover catalysis of RNA cleavage by bulge–loop inducing supramolecular conjugates

Amirloo

Staroseletz

Yousaf

et al. 2021

Nucleic Acids Research

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“…Cas12g was characterized by RNA-guided collateral ribonuclease and single-strand deoxyribonuclease activities ( Yan et al, 2019 ). Cas12f (also known as Cas14) nucleases cleave single- and dsDNA targets triggered by a 5′ T- or C-rich PAM sequence ( Harrington et al, 2018 ; Karvelis et al, 2020 ; Bigelyte et al, 2021 ; Xu X. et al, 2021 ; Takeda et al, 2021 ).…”

Section: Crispr-based Biosensing Techniquesmentioning

confidence: 99%

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

Chen

et al. 2022

Front. Bioeng. Biotechnol.

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In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.

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“…Obligated mobile element-guided activity (OMEGA) is another class of transposon-encoded RGENs with a reduced size (∼400 amino acids) ( Altae-Tran et al, 2021 ). Miniature CRISPR associated RGENs such as Cas12f are also being explored for gene editing capability and engineered for improved efficiency ( Bigelyte et al, 2021 ; Xu et al, 2021 ). The discovery and optimization of these smaller genome editing tools may facilitate delivery with germline infecting PSVs such as TRV or BSMV.…”

Section: Virus Induced Genome Editingmentioning

confidence: 99%

Non-GM Genome Editing Approaches in Crops

2021

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CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells

Cited by 63 publications

References 49 publications

“Bind, cleave and leave”: multiple turnover catalysis of RNA cleavage by bulge–loop inducing supramolecular conjugates

“Bind, cleave and leave”: multiple turnover catalysis of RNA cleavage by bulge–loop inducing supramolecular conjugates

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

Non-GM Genome Editing Approaches in Crops

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