Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

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Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

Nucleic Acids Research ◽

10.1093/nar/gkz1136 ◽

2019 ◽

Vol 48 (2) ◽

pp. 517-532 ◽

Cited By ~ 14

Author(s):

Bin Liu ◽

Siwei Chen ◽

Anouk La Rose ◽

Deng Chen ◽

Fangyuan Cao ◽

...

Keyword(s):

Gene Editing ◽

Gene Knockout ◽

Rapid Development ◽

Open Chromatin ◽

Chromatin Compaction ◽

Histone Deacetylase 1 ◽

Cas9 Protein ◽

Target Dna ◽

Histone Modifiers ◽

Low Efficiency

Abstract Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

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CRISPR/Cas9 gene editing: Applications in Parasitology

QJM ◽

10.1093/qjmed/hcaa060.009 ◽

2020 ◽

Vol 113 (Supplement_1) ◽

Author(s):

K E Khalifa

Keyword(s):

Cancer Research ◽

Gene Editing ◽

Disease Models ◽

Research Development ◽

Gene Drive ◽

Water Flea ◽

Knock Out ◽

Cas9 Protein ◽

Target Dna ◽

Made In

Abstract Clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) technology is adapted from the bacterial immune system and has rapidly revolutionized the editing of many genomes. This system is based on two components: an RNA-guided endonuclease, classically the Cas9 protein, and an RNA molecule that guides (gRNA) the nuclease to a complementary locus in the target DNA. The technique has allowed researchers to do gene knock-out, knock-in, insertions and deletions in cell lines and animals in rapid efficient way. The technique has many applications in the study of genes function, gene drive, cancer research, development of disease models and new therapeutic targets and crops enhancement. In the field of parasitology, using CRISPR/Cas9, significant advancements have been made in genome editing of Plasmodium spp, Leishmania, Typanosoma cruzi, and Toxoplasma gondii, as well as vectors like the mosquito, water flea and ticks. The aim of the talk is to present the basics of CRISPR/Cas9 and summarize some of its applications in the field of parasitology

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One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

International Journal of Molecular Sciences ◽

10.3390/ijms22052249 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2249

Author(s):

Fuminori Tanihara ◽

Maki Hirata ◽

Nhien Thi Nguyen ◽

Osamu Sawamoto ◽

Takeshi Kikuchi ◽

...

Keyword(s):

Gene Editing ◽

Multiple Gene ◽

Knock Out ◽

Editing Efficiency ◽

Guide Rnas ◽

Cas9 Protein ◽

One Step ◽

Step Generation ◽

The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

10.1101/670554 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bin Liu ◽

Siwei Chen ◽

Anouk La Rose ◽

Deng Chen ◽

Fangyuan Cao ◽

...

Keyword(s):

Gene Editing ◽

Gene Knockout ◽

Rapid Development ◽

Open Chromatin ◽

Chromatin Compaction ◽

Histone Deacetylase 1 ◽

Cas9 Protein ◽

Target Dna ◽

Histone Modifiers ◽

Low Efficiency

AbstractDespite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

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Lentivirus pre-packed with Cas9 protein for safer gene editing

Gene Therapy ◽

10.1038/gt.2016.27 ◽

2016 ◽

Vol 23 (7) ◽

pp. 627-633 ◽

Cited By ~ 61

Author(s):

J G Choi ◽

Y Dang ◽

S Abraham ◽

H Ma ◽

J Zhang ◽

...

Keyword(s):

Gene Editing ◽

Cas9 Protein

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Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs

Methods in Molecular Biology - CRISPR Gene Editing ◽

10.1007/978-1-4939-9170-9_9 ◽

2019 ◽

pp. 127-134 ◽

Cited By ~ 2

Author(s):

Anders Laustsen ◽

Rasmus O. Bak

Keyword(s):

Gene Editing ◽

Chemically Modified ◽

Cas9 Protein

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574. Lentivirus Pre-Packed with Cas9 Protein for Safer Gene Editing

Molecular Therapy ◽

10.1016/s1525-0016(16)33382-2 ◽

2016 ◽

Vol 24 ◽

pp. S229

Author(s):

Manjunath Swamy ◽

Jang-Gi Choi ◽

Ying Dang ◽

Sojan Abraham ◽

Hongming Ma ◽

...

Keyword(s):

Gene Editing ◽

Cas9 Protein

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A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

10.20944/preprints201811.0159.v1 ◽

2018 ◽

Author(s):

Zhanqi Dong ◽

Qi Qin ◽

Zhigang Hu ◽

Peng Chen ◽

Liang Huang ◽

...

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

Sustained Delivery ◽

Dna Breaks ◽

Guide Rna ◽

Cas9 Protein ◽

Transgenic Silkworms ◽

Enzyme Targeting ◽

Multiplex System ◽

Unique Capability

Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

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