CRISPR/Cas9‐Directed Genome Editing of Cultured Cells

Yang, Luhan; Yang, Jason; Byrne, Susan; Pan, Joshua; Church, George M.

doi:10.1002/0471142727.mb3101s107

Cited by 76 publications

(72 citation statements)

References 43 publications

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“…CRISPR/Cas9 genome modification is a powerful approach for generating both targeted indel mutations by non-homologous endjoining (NHEJ) repair and more precise gene editing by the introduction of specific sequences through homology-directed repair (HDR) (Auer et al, 2014;Bassett et al, 2014;Bhattacharya et al, 2015;Blitz et al, 2013;Chen et al, 2013;Friedland et al, 2013;Guo et al, 2014;Hisano et al, 2015;Kimura et al, 2014;Kotani et al, 2015;Li et al, 2015;Nakayama et al, 2013Nakayama et al, , 2014Ran et al, 2013;Shi et al, 2015;Wang et al, 2015;Yang et al, 2014). While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging.…”

Section: Introductionmentioning

confidence: 99%

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

et al. 2017

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The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

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Section: Introductionmentioning

confidence: 99%

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

et al. 2017

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“…It is derived from type II bacterial adaptive immune system. The CRISPR-Cas9 mediated immunity is explicitly explained in Yang et al [128]. The hallmark of this system is the short RNA guide that recognizes the target DNA through base pairing, forming a RNA-DNA complex, subsequently Cas9 creates DSB on the target sequence [129,130] and a designed DNA fragment can be specifically incorporated.…”

Section: In Vivo Approaches Towards Affinity Maturation Of Antibodiesmentioning

confidence: 99%

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

Lim¹,

Choong²,

Lim³

2018

Antibody Engineering

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“…25 HEK293T cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 4 mmol/L l-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C and 5% CO 2 in a humidified incubator. HEK293T cells were seeded in 6-well plates 24 hours before transfection and were ≈40% confluent at transfection.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…To precisely validate the effect of the hypertension-associated SNP (rs3754777) in the STK39 gene on biochemical function in cultured cells, we generated knockin cell lines carrying SNP (A>G) in the STK39 gene using the CRISPR/Cas9-mediated genome-editing system (24)(25)(26). In this system, we prepared a double-nickase (Cas9-D10A) plasmid and sgRNA plasmids for the double nicking-targeted sites flanking SNP and ssODN that provide a mutated allele sequence.…”

Section: Generation Of the 2 Hek293t Cell Lines Carrying The Homozygomentioning

confidence: 99%

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System

et al. 2015

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Abstract-Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, singleguide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/ SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism.

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CRISPR/Cas9‐Directed Genome Editing of Cultured Cells

Cited by 76 publications

References 43 publications

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System

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