Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

DiNapoli, Sara; Martinez-McFaline, Raúl; Gribbin, Caitlin; Wrighton, Paul J.; Balgobin, Courtney A.; Nelson, Isabel; Leonard, Abigail J.; Maskin, Carolyn; Shwartz, Arkadi; Quenzer, Eleanor D.; Mailhiot, Darya; Kao, Clara; McConnell, Sean C.; Jong, Jill L. O. de; Goessling, Wolfram; Houvras, Yariv

doi:10.1093/nar/gkaa085

Cited by 36 publications

(30 citation statements)

References 34 publications

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“…In our knock-in study, we employed CRISPR-Cas9 RNPs that had been assembled in vitro using two synthetic RNA oligonucleotides (a target-specific crRNA and a universal tracrRNA) and the Cas9 protein to introduce DSBs. Recent reports demonstrated that the same crRNA/tracrRNA-based RNP system is highly mutagenic in zebrafish embryos ( Hoshijima et al, 2019 ; DiNapoli et al, 2020 ). RNP-injected embryos often show null mutant phenotypes ( Hoshijima et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

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

Ranawakage

Okada

Sugio

et al. 2021

Front. Cell Dev. Biol.

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

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Section: Discussionmentioning

confidence: 99%

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

Ranawakage

Okada

Sugio

et al. 2021

Front. Cell Dev. Biol.

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“…This doesn't mean we can't use it for dsDNA. By targeting both dsDNA (DiNapoli et al, 2020[ 29 ]) and ssDNA (Bai et al, 2020[ 12 ]) templates in cells, experimental animals (Pineault et al, 2019[ 102 ]), plants (Mao et al, 2019[ 87 ]), model organisms (Bai et al, 2020[ 11 ]) and even humans (Ma et al, 2015[ 79 ]; Moon et al, 2019[ 91 ]), we can regulate insertions, deletions, replication, migration, transformation, copy number loss or gain (Anzalone et al, 2019[ 6 ]) with the CRISPR system. Different Cas enzymes are preferred according to the mechanisms by which they are effective in all these biological structures.…”

Section: Mechanisms Of Genome Editing Toolsmentioning

confidence: 99%

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

Yılmaz

2021

EXCLI Journal; 20:Doc19; ISSN 1611-2156

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“…Zebrafish embryos are amenable to genetic engineering and have been extensively used as a vertebrate proving ground for novel genome altering tools. 21 –24 The application of those tools in zebrafish has vastly broadened our understanding of basic vertebrate biology and human diseases in diverse areas such as metabolic, muscular, skeletal, neuronal, cardiovascular, and cancer. 25 –31 Larvae are transparent, facilitating in vivo examination of internal structures, and can survive for several days post-fertilization in just a drop of water.…”

Section: General Advantages and Challenges Of Zebrafish As An Animal mentioning

confidence: 99%

Zebrafish as an Animal Model for Ocular Toxicity Testing: A Review of Ocular Anatomy and Functional Assays

2020

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Xenobiotics make their way into organisms from diverse sources including diet, medication, and pollution. Our understanding of ocular toxicities from xenobiotics in humans, livestock, and wildlife is growing thanks to laboratory animal models. Anatomy and physiology are conserved among vertebrate eyes, and studies with common mammalian preclinical species (rodent, dog) can predict human ocular toxicity. However, since the eye is susceptible to toxicities that may not involve a histological correlate, and these species rely heavily on smell and hearing to navigate their world, discovering visual deficits can be challenging with traditional animal models. Alternative models capable of identifying functional impacts on vision and requiring minimal amounts of chemical are valuable assets to toxicology. Human and zebrafish eyes are anatomically and functionally similar, and it has been reported that several common human ocular toxicants cause comparable toxicity in zebrafish. Vision develops rapidly in zebrafish; the tiny larvae rely on visual cues as early as 4 days, and behavioral responses to those cues can be monitored in high-throughput fashion. This article describes the comparative anatomy of the zebrafish eye, the notable differences from the mammalian eye, and presents practical applications of this underutilized model for assessment of ocular toxicity.

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Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

Cited by 36 publications

References 34 publications

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

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

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

Zebrafish as an Animal Model for Ocular Toxicity Testing: A Review of Ocular Anatomy and Functional Assays

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