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The advances in CRISPR technology and 3D genome.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is a prokaryotic immune system that used to resist foreign genetic factors. It rapidly becomes the hot technology in life sciences and is applies for genome editing to solve the problem of genome-derived diseases. Using CRISPR/Cas technique, the biological DNA sequence can be repaired, cut, replaced, or added. It can effectively change the human stem cells and is expected to achieve results in the treatment. Compared with ZFN and TALEN genome editing techniques, CRISPR is more effective, accurate, and convenient. The application of CRISPR technique in three dimensional (3D) genome structure makes us understand the relationship between linear DNA sequence and 3D chromatin structure. Utilizing CRISPR/Cas9 genome editing to reverse or delete CTCF binding sites, to recognize changes of topological isomerism of the genome and interactions between chromatin loops. The purpose of this review is to introduce the characteristics and classification of the current CRISPR/Cas system, multiple functions, and potential therapeutic uses, as well as to outline the effect of the technique on chromatin loops by changing CTCF sites in 3D genomes. We will also briefly describe the importance of ethical dilemmas to be faced in CRISPR applications and provide a perspective for potential CRISPR considerations.
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CRISPR/Cas9-mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease.
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In utero gene editing as a treatment for heritable metabolic syndromes.
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Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.
Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.
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Therapeutic and diagnostic relevance of Crispr technology.
CRISPR is a family of DNA repeats providing immunity against viral and plasmid invading DNA in bacteria and archaea. The system consist of an endonuclease Cas, guided by a RNA sequence, able to cleave the DNA double strand at a specific site. The discovery of Crispr function in 2007 has revolutionized genetic engineering by giving to the world the most powerful and precise tool for targeted genome editing. The aim of this review is to synthesize the current knowledge on Crispr/cas system and its application in biomedical field. In particular, we focus on the relevance of this new tool in progressing our comprehension for biological mechanisms and improving our ability to treat and prevent genetic diseases, to control microbial virulence and to generate animal models for basic and clinical research. We discuss also the ethical issues that may prevent the application of Crispr technology in living beings.
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Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.
Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)-mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR-mediated GT is extremely rare in higher plants, positive-negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac-transposon-mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I-SceI break and subsequent single-strand annealing (SSA)-mediated DNA repair system.
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[Genome editing ~Principle and possibility of a novel genetic engineering technology. Basic principles of genome editing.].
Programmable site-specific nuclease mediated-genome editing is an emerging biotechnology for precise manipulation of target genes. In genome editing, gene-knockout as well as gene-knockin are possible in various organisms and cultured cells. CRISPR-Cas9, which was developed in 2012, is a convenient and efficient programmable site-specific nuclease and the use spreads around the world rapidly. For this, it is important for the progress of life science research to introduce the genome editing technology.
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CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.
Discovered as a bacterial adaptive immune system, CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR associated) is being developed as an attractive tool in genome editing. Due to its high specificity and applicability, CRISPR/Cas9-mediated gene editing has been employed in a multitude of organisms and cells, including insects, for not only fundamental research such as gene function studies, but also applied research such as modification of organisms of economic importance. Despite the rapid increase in the use of CRISPR in insect genome editing, results still differ from each study, principally due to existing differences in experimental parameters, such as the Cas9 and guide RNA form, the delivery method, the target gene and off-target effects. Here, we review current reports on the successes of CRISPR/Cas9 applications in diverse insects and insect cells. We furthermore summarize several best practices to give a useful checklist of CRISPR/Cas9 experimental setup in insects for beginners. Lastly, we discuss the biosafety concerns related to the release of CRISPR/Cas9-edited insects into the environment.
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Reply to "CRISPR screens are feasible in TP53 wild-type cells".
Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups.
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Geminiviral-induced genome editing using miniature CRISPR/Cas12j (CasΦ) and Cas12f variants in plants.
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