Gene Targeting Through Homologous Recombination in Monkey Embryonic Stem Cells Using CRISPR/Cas9 System

Zhu, Sipin; Rong, Zhili; Lu, Xiaoyan; Fu, Xuemei

doi:10.1089/scd.2014.0507

Cited by 8 publications

(9 citation statements)

References 7 publications

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“…21 It was confirmed in another study where authors performed targeted genome editing in a single rhesus ESC line at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. 20 This is the first study reporting successful CRISPR/Cas9-mediated gene addition at the AAVS1 safe harbor locus in NHP PSCs. Modified cells retained their pluripotent phenotype and showed no abnormal karyotype.…”

Section: Discussionmentioning

confidence: 94%

“…[16][17][18] Recent reports have indicated that the CRISPR/Cas9 system can be used for genetic modification in NHPs; however, this approach has not yet been applied to insert genes of interest into the AAVS1 safe harbor in NHP PSCs. [19][20][21] We have identified a CRISPR/Cas9 target sequence in the rhesus macaque PPP1R12C gene, generated safe harbor-targeted rhesus macaque iPSC (RhiPSC) lines with two relevant marker genes, and documented the stability of expression both in iPSCs and after differentiation to tissues of interest. Genes encoding human truncated CD19 (hDCD19) or GFP were chosen as reporters.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

et al. 2017

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Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

et al. 2017

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“…In conclusion, our SSA‐mediated scarless gene precise editing method does not leave any scar within the genome in contrast to the Cre/LoxP strategy and avoids the risk of reintegration of the piggyBac transposon as the CRISPR/Cas9‐ piggyBac method , therefore providing an important tool for the future seamless genome editing and gene correction.…”

Section: Discussionmentioning

confidence: 97%

“…However, the marker gene remained in the genome may suppress the allele transcription or unintentionally disrupt gene splicing . The Cre/LoxP system has been used for removing marker genes, which is known as marker‐free but actually leaves behind a 34‐base pair of loxP sequence along with the targeted mutation . This small ectopic sequence has the potential to affect the expression of adjacent genes in mammalian cells .…”

Section: Introductionmentioning

confidence: 99%

Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

et al. 2018

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CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34-base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing. Here, we report a novel two-step precise gene-editing method by leveraging the SSA-mediated repair mechanism into the CRISPR/Cas9-mediated gene-editing system. An integrating cassette was developed with positive and negative selection markers, which was flanked by direct repeat sequences with desired mutations as SSA arms. After the targeted integration of the cassette mediated by CRISPR/Cas9-induced homologous-directed repair, cell clones were first selected through the positive selection. In the second round targeting, the selection cassette was removed by the SSA-mediated DNA double-strand break (DSB) repair without any scar left behind. The novel seamless genome editing technique was tested on CCR5 and APP loci, and finally demonstrated, respectively, up to 45.83% and 68% of precise genome editing efficiency. This study provides a new efficient approach for precise genome editing and gene correction.

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“…In previously used Cre/Lox P system-mediated gene targeting strategies, the removal of selection cassettes presumably left residual sequences in the targeted loci, which may lead to detrimental effects in cells. For instance, the Cre/Lox P system leaves a 34-bp Lox P sequence when releasing the insertion 38 , 39 . However, our new strategy using the piggyBac construct, flanked by homologous genomic sequences, could seamlessly remove the selection cassette and reintegrate the P191A mutation into the CXCR4 gene instead of introducing of exogenous sequences in the recombinant allele.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 inhibition in cells with CXCR4 mutant genome created by CRISPR-Cas9 and piggyBac recombinant technologies

Shuai

Wang

et al. 2018

Sci Rep

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The C-X-C chemokine receptor type 4 (CXCR4) is one of the major co-receptors for human immunodeficiency virus type 1 (HIV-1) entry and is considered an important therapeutic target. However, its function in maintaining the development of hematopoietic stem cells (HSC) makes it difficult to be used for HIV-1 gene therapy with HSC transplantation. A previous report showed that the natural CXCR4 P191A mutant inhibits HIV-1 infection without any defect in HSC differentiation, which could provide a basis for the development of new approaches for HIV-1 gene therapy. In the present study, we used CRISPR-Cas9 combined with the piggyBac transposon technologies to efficiently induce the expression of the CXCR4 P191A mutant in an HIV-1 reporter cell line, leading to no detectable exogenous sequences. In addition, no off-target effects were detected in the genome-edited cells. The decline of HIV-1 replication in biallelic CXCR4 gene-edited cells suggests that individuals equipped with homologous recombination of the CXCR4 P191A mutant could prevent or reduce HIV-1 infection. This study provides an effective approach to create a CXCR4 mutation with HIV-1 infection inhibition function and without leaving any genetic footprint inside cells, thereby shedding light on an application in HIV-1 gene therapy and avoiding side effects caused by deficiency or destruction of CXCR4 function.

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Gene Targeting Through Homologous Recombination in Monkey Embryonic Stem Cells Using CRISPR/Cas9 System

Cited by 8 publications

References 7 publications

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

HIV-1 inhibition in cells with CXCR4 mutant genome created by CRISPR-Cas9 and piggyBac recombinant technologies

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