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Abstract Background Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

Background: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells. [ABSTRACT FROM AUTHOR]

Abstract We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5′-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRISPR-Cas9 genome editing technology has been successfully applied to generate various genetic modifications in zebrafish. The CRISPR-Cas9 system, which originally consisted of three components, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9, efficiently induces DNA double-strand breaks (DSBs) at targeted genomic loci, often resulting in frameshift-mediated target gene disruption (knockout). However, it remains difficult to perform the targeted integration of exogenous DNA fragments (knock-in) with CRISPR-Cas9. DSBs can be restored through DNA repair mechanisms, such as nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). One of our two research groups established a method for the precise MMEJ-mediated targeted integrations of exogenous genes containing homologous microhomology sequences flanking a targeted genomic locus in zebrafish. The other group recently developed a method for knocking in ~200 nt sequences encoding composite tags using long single-stranded DNA (ssDNA) donors. This chapter summarizes the CRISPR-Cas9-mediated genome modification strategy in zebrafish., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A colorimetric sensor based on gold nanoparticles (AuNPs) and single-stranded DNA (ssDNA) is a simple and rapid method for detecting foodborne pathogens. However, the colorimetric method employed in previous studies involved short ssDNA (<100 nucleotides), including the aptamer and PCR products, resulting in the high detection limit of this technique. In this study, a colorimetric sensor was developed based on long ssDNA of asymmetric PCR (aPCR) and non-functionalized AuNPs for detecting Salmonella Typhimurium (S. Typhimurium). In the presence of S. Typhimurium, the long ssDNA (547 nt) amplified by aPCR-protected AuNPs from NaCl-induced aggregation, while the solution retained a red color. After optimizing parameters, the limit of detection (LOD) of the colorimetric sensor was 2.56 CFU/mL with high specificity. Recovery studies showed its feasibility for detecting S. Typhimurium (102 CFU/mL, 104 CFU/mL, and 106 CFU/mL) in spiked lettuce samples. This colorimetric sensor provides new opportunities for the highly sensitive detection of bacteria in real food samples. [Display omitted] • Colorimetric method based on long ssDNA and non-functionalized gold nanoparticles. • Long ssDNA could be obtained by aPCR-protected AuNPs from NaCl-induced aggregation. • The limit of detection was 2.56 CFU/mL. • Recovery rate of S. Typhimurium in s piked lettuce ranged from 91.84% to 110.2%. [ABSTRACT FROM AUTHOR]

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5′-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research. [ABSTRACT FROM AUTHOR]

Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).