A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing

Theranostics, Год журнала: 2020, Номер 10(12), С. 5532 - 5549

Опубликована: Янв. 1, 2020

Gene editing is a versatile technique in biomedicine that promotes fundamental research as well clinical therapy.The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome machinery has accelerated the application gene editing.However, delivery CRISPR components often suffers when using conventional transfection methods, such viral transduction and chemical vectors, due to limited packaging size inefficiency toward certain cell types.In this review, we discuss physical methods for which can overcome these limitations.We outline different types highlight novel techniques deliver components, emphasize role micro nanotechnology improve performance.We present our perspectives on limitations current technology provide insights future developments methods.

Язык: Английский

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CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection

Cell Reports, Год журнала: 2019, Номер 27(13), С. 3780 - 3789.e4

Опубликована: Июнь 1, 2019

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and replacement; however, existing methods to generate such animals remain laborious costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR delivery with Cas9/sgRNA engineer in the genome high efficiency throughput. We successfully targeted 774 bp fluorescent reporter, 2.1 kb CreERT2 driver, 3.3 expression cassette into loci both embryos live mice. is applicable most widely used knockin schemes requiring lengths within 4.9 packaging capacity. Altogether, efficient, high-throughput, microinjection-free sophisticated engineering potential other mammalian species.

Язык: Английский

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Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

Science Advances, Год журнала: 2020, Номер 6(7)

Опубликована: Фев. 13, 2020

Knock-in genome targeting risks: Comprehensive locus analysis is essential for precision chromosome-editing identification.

Язык: Английский

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Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

Genome biology, Год журнала: 2019, Номер 20(1)

Опубликована: Авг. 25, 2019

CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up 16% efficiency in generating conditional (cKO or floxed) alleles by microinjection 2 single guide RNAs (sgRNA) single-stranded oligonucleotides as donors (referred herein "two-donor floxing" method).We re-evaluate two-donor method from a consortium 20 laboratories across world. The dataset constitutes 56 genetic loci, 17,887 zygotes, 1718 live-born which only 15 (0.87%) mice contain cKO alleles. We subject statistical analyses machine learning algorithm, reveals that none factors analyzed was predictive for success this method. test some newer methods use one-donor DNA on 18 loci approach failed produce find are 10- 20-fold more efficient than approach.We propose lacks because it relies two simultaneous recombination events cis, outcome is dwarfed pervasive accompanying undesired editing events. fairly they rely one event, probability correct insertion donor cassette without unanticipated mutational much higher. Therefore, offer higher efficiencies routine animal models.

Язык: Английский

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Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

iScience, Год журнала: 2018, Номер 9, С. 286 - 297

Опубликована: Ноя. 1, 2018

Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo have been established only a small set animals. To overcome these issues, we developed method large-fragment DNA knockin micromanipulation. In this study, successfully delivered the donor into zygotes adeno-associated virus (AAV) removing zona pellucida, and succeeded both large-DNA fragment whole exon exchange with electroporation ribonucleoprotein. By method, can fragments conveniently various animal species

Язык: Английский

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Principles of Genetic Engineering

Genes, Год журнала: 2020, Номер 11(3), С. 291 - 291

Опубликована: Март 10, 2020

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety approaches. For example, homologous recombination can be used target specific sequences mouse embryonic stem (ES) cell genomes or other cultured cells, but it cumbersome, poorly efficient, and relies on drug positive/negative selection culture for success. Other routinely applied methods include random integration after direct transfection (microinjection), transposon-mediated insertion, insertion mediated by viral vectors production transgenic mice rats. Random occurs more frequently than recombination, has numerous drawbacks, despite its efficiency. The most elegant effective method based guided endonucleases, because these sequences. Since advent clustered regularly interspaced short palindromic repeats CRISPR/Cas9 technology, endonuclease-mediated gene targeting become widely engineer supplanting zinc finger nucleases, transcription activator-like effector meganucleases. Future improvements editing may achieved increasing efficiency homology-directed repair. Here, we describe principles genetic detail: (1) how common elements current technologies need chromosome break occur, (2) sensitive genotyping assays detect altered (3) delivery modalities that impact characterization modifications. In summary, while some remain steadfast, others change as are ever-evolving continue revolutionize research many fields.

Язык: Английский

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Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease

Nature Communications, Год журнала: 2019, Номер 10(1)

Опубликована: Дек. 11, 2019

Abstract Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical phenotypes, including onset heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD with cynomolgus monkeys. As humans mice, near-complete depletion induces severe formation mainly collecting ducts. Importantly, unlike heterozygote monkeys exhibit perinatally distal tubules, possibly reflecting initial pathology humans. Many these survive after formation, cysts progress age. Furthermore, succeed generating selective heterozygous allele-specific targeting. We propose that our elucidate progression ADPKD, which will serve as a basis for establishing new therapeutic strategies, drug treatments.

Язык: Английский

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CRISPR-Based Tools in Immunity

Annual Review of Immunology, Год журнала: 2019, Номер 37(1), С. 571 - 597

Опубликована: Янв. 30, 2019

CRISPR technology has opened a new era of genome interrogation and engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the system been repurposed as an adaptable tool for editing multiple other applications. CRISPR's ease use, precision, versatility have led to its widespread adoption, accelerating biomedical research discovery human cells model organisms. Here we review CRISPR-based tools discuss how they are being applied decode genetic circuits that control immune function health disease. Genetic variation can affect autoimmune disease risk, infectious pathogenesis, cancer immunotherapies. provides unprecedented opportunities functional mechanistic studies coding noncoding sequence immunity. Finally, potential engineer synthetic cellular immunotherapies wide range diseases.

Язык: Английский

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Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

Scientific Reports, Год журнала: 2020, Номер 10(1)

Опубликована: Окт. 21, 2020

Abstract CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for development of genetically modified mouse models. In recent years, number reports have demonstrated effective delivery via electroporation an alternative conventional method microinjection. this study, we performed side-by-side comparisons two RNP methods across multiple gene loci and conclude that compares very favourably with pronuclear microinjection, report improvement in mutagenesis efficiency when delivering CRISPR generation simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. addition, show can be further increased by embryos derived from Cas9-expressing donor females. The maternal supply Cas9 avoids necessity deliver relatively large protein, high both indel allele achieved small single-guide RNAs ssODN templates alone. Furthermore, electroporation, compared results higher rate embryo survival development. thus potential reduce animals used production

Язык: Английский

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Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor

Frontiers in Cell and Developmental Biology, Год журнала: 2021, Номер 8

Опубликована: Фев. 11, 2021

Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, efficiency and precision this technology still require further optimization, particularly for multicellular model organisms, such as zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into genome using combination CRISPR-Cas9 ribonucleoprotein complex long single-stranded DNA (lssDNA) donor template. Here, we sox3, sox11a, pax6a genes to evaluate knock-in lssDNA donors with different structures in somatic cells injected embryos their germline transmission. The characteristics templates were found crucial achieve high rate precise heritable knock-ins. following our key findings: (1) strand selection is important; however, preference its dependency appear vary among target loci or sequences. (2) length 3' homology arm affects site-specific manner; particularly, shorter 50-nt leads higher than longer 300-nt sox3 (3) Some distance between cleavage site insertion adversely affect repair process, resulting imprecise editing. By implementing proposed method, successfully obtained precisely edited alleles contained composed FLAGx3 (or PAx3), Bio tag, HiBiT His tag) moderate transmission rates 21%. Furthermore, allele-specific quantitative polymerase chain reaction (qPCR) both 5' junctions indicated allele frequencies at side lssDNAs, suggesting lssDNA-templated was mediated by unidirectional single-strand template (SSTR) embryos.

Язык: Английский

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