Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

Wang, Kankan; Tang, Xiaochun; Liu, Yan; Xie, Zhixun; Zou, Xiaodong; Li, Mengjing; Yuan, Hongming; Ouyang, Hongsheng; Jiao, Huping; Pang, Daxin

doi:10.1038/mtna.2016.101

Cited by 40 publications

(30 citation statements)

References 53 publications

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“…It has been shown in human cells that NGA PAMs may have up to 40% activity in some loci 74 , 75 . Interestingly, we also observed that some clones contained the 535A > T substitution on only one allele while the PAM mutation was present on the two alleles suggestive of partial or incomplete HDR and has been reported by others 27 , 31 , 35 . This may be indicative of a cut-to-distance dependence mechanism in the incorporation of single nucleotide edits in chicken PGCs as previously reported in human cells and mouse zygotes 27 , 35 .…”

Section: Discussionsupporting

confidence: 86%

High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

Idoko-Akoh

Taylor

Sang

et al. 2018

Sci Rep

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Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

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Section: Discussionsupporting

confidence: 86%

High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

Idoko-Akoh

Taylor

Sang

et al. 2018

Sci Rep

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“…The RAD51 stimulatory compound RS-1 increased knock-in efficiency in rabbit embryos (from 4.4 to 26.1%) 18 , HEK cells (from 3.5 to 21%) 17 and U2OS cells (from 1.9 to 2.4%) 17 , but not in hiPSCs 19 . No effect of RS-1 on TNS efficiency was found in porcine fetal fibroblasts 23 . Furthermore, We tested these molecules in iPSC lines that we generated that carry doxycycline-inducible Cas9 (iCRISPR-Cas9) and Cas9 nickase with the D10A mutation (iCRISPR-Cas9n) integrated in their genomes 11 .…”

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confidence: 83%

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Riesenberg

Maričić

2017

Preprint

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A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks (DSBs) which are then repaired by homology-directed repair (HDR) using synthetic or cloned DNA donor molecules carrying desired mutations. However, another pathway, the non-homologous end joining (NHEJ) pathway competes with HDR for repairing DNA breaks in cells. To increase the frequency of precise genome editing in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) we have tested the capacity of a number of small molecules to enhance HDR or inhibit NHEJ. We identify molecules that increase the frequency of precise genome editing including some that have additive effects when applied together. Using a mixture of such molecules, the 'CRISPY' mix, we achieve 2.8-to 6.7-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes, to our knowledge the highest editing efficiency in hiPSCs described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.9-to 4.0-fold, allowing almost 20% of chromosomes to be edited. Main textThe bacterial nuclease CRISPR/Cas9 is now frequently used to accurately cut chromosomal DNA sequences in eukaryotic cells. The resulting DNA breaks are repaired by two competing pathways: NHEJ and HDR (Fig. 1). In NHEJ, the first proteins to bind the cut DNA ends are Ku70/Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) Since NHEJ of Cas9-induced DSBs is error prone and frequently introduces short insertions and deletions (indels) at the cut site, it is useful for knocking out a targeted gene. In contrast, HDR allows precise repair of a DSB by using a homologous donor DNA. If the donor DNA provided in the experiment carries mutations, these will be introduced into the genome (precise genome editing). Repair with homologous ssDNA or dsDNA has been suggested to engage different pathways 5 . We will refer to Targeted Nucleotide Substitutions using ssDNA donors as 'TNS' and targeted insertion of cassettes . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/136374 doi: bioRxiv preprint first posted online May. 10, 2017; 3 using dsDNA donors as knock-ins, respectively. In order to introduce a DSB Cas9 requires the nucleotide sequence NGG (a "PAM" site) in the target DNA. Targeting of Cas9 is further determined by a guide RNA (gRNA) complementary to 20 nucleotides adjacent to the PAM site. However, the Cas9 may also cut the genome at sites that carry sequence similarity to the gRNA 6 . One strategy to reduce such off-target cuts is to use a mutated Cas9 that introduces single-stranded nicks instead of DSBs (Cas9n) 7 . Using two gRNAs to introduce two nicks on opposite DNA strands in close proximity to each other will result in a staggered D...

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“…Genome editing conducted by this mechanism can be used to insert a predefined single nucleotide or sequence, or even change or delete it in existing genes. After the first knockouts births reported by NHEJ, successful generation of knock-in large animals by HDR were achieved in sheep [15,16], goats [17] and pigs [18].…”

Section: Introductionmentioning

confidence: 99%

CRISPR in livestock: From editing to printing

et al. 2020

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a b s t r a c tPrecise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time-and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.

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Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

Cited by 40 publications

References 53 publications

High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

CRISPR in livestock: From editing to printing

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