Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293FT cells

Published on Dec 1, 2016in SpringerPlus
· DOI :10.1186/s40064-016-2536-3
Zuyong He13
Estimated H-index: 13
(Edin.: University of Edinburgh),
Christopher Proudfoot11
Estimated H-index: 11
(Edin.: University of Edinburgh)
+ 1 AuthorsSimon G. Lillico24
Estimated H-index: 24
(Edin.: University of Edinburgh)
Background Genome editors such as CRISPR/Cas9 and TALENs are at the forefront of research into methodologies for targeted modification of the mammalian genome. To date few comparative studies have been carried out to investigate the difference of genome editing characteristics between CRISPR/Cas9 and TALENs. While the CRISPR/Cas9 system has overtaken TALENs as the tool of choice for most research groups working in this field, we hypothesized that there could be certain applications whereby the application of TALENs would have specific benefits. Here we compare CRISPR/Cas9 and TALEN as tools for introducing site-specific editing events at an integrated EGFP gene in the genome of HEK293FT cells.
  • References (24)
  • Citations (13)
📖 Papers frequently viewed together
1 Author (Chao Feng)
5,693 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1Meilin ZhuH-Index: 1
111 Citations
A powerful gene-editing technology is the biggest game changer to hit biology since PCR. But with its huge potential come pressing concerns.
170 CitationsSource
#1Michael V. WilesH-Index: 1
#2Wenning QinH-Index: 4
Last. Haoyi Wang (CAS: Chinese Academy of Sciences)H-Index: 26
view all 4 authors...
CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR–Cas9 system holds promise as a “system of choice” for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRISPR–...
27 CitationsSource
#1Zuyong He (The Roslin Institute)H-Index: 13
#2Christopher Proudfoot (Edin.: University of Edinburgh)H-Index: 11
Last. Simon G. Lillico (Edin.: University of Edinburgh)H-Index: 24
view all 6 authors...
The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervenin...
36 CitationsSource
#1ChongHua RenQiang (NWAFU: Northwest A&F University)H-Index: 10
#2Kun Xu (NWAFU: Northwest A&F University)H-Index: 17
Last. Zhiying Zhang (NWAFU: Northwest A&F University)H-Index: 12
view all 7 authors...
Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair s...
19 CitationsSource
#1Simon G. Lillico (Edin.: University of Edinburgh)H-Index: 24
#2Christopher Proudfoot (Edin.: University of Edinburgh)H-Index: 11
Last. C. Bruce A. Whitelaw (Edin.: University of Edinburgh)H-Index: 19
view all 13 authors...
Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any...
114 CitationsSource
#1Patrick HsuH-Index: 19
#2Douglas ScottH-Index: 111
Last. Feng ZhangH-Index: 118
view all 14 authors...
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.
2,285 CitationsSource
#1F. Ann RanH-Index: 8
#2Patrick HsuH-Index: 19
Last. Feng ZhangH-Index: 118
view all 11 authors...
Summary Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in ...
1,918 CitationsSource
#1An Xiao (UCLA: University of California, Los Angeles)H-Index: 9
#2Zhanxiang Wang (UCLA: University of California, Los Angeles)H-Index: 3
Last. Bo Zhang (UCLA: University of California, Los Angeles)H-Index: 8
view all 13 authors...
Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic de...
293 CitationsSource
#1Yung-Tae Kim (SNU: Seoul National University)H-Index: 3
#2Jiyeon Kweon (SNU: Seoul National University)H-Index: 10
Last. Jin-Soo Kim (SNU: Seoul National University)H-Index: 58
view all 19 authors...
A collection of TALENs targeted to 18,740 human protein-coding genes will facilitate genetic engineering of human cells.
266 CitationsSource
Cited By13
#1Sara Mata López (A&M: Texas A&M University)H-Index: 4
#2Cynthia J. Balog-Alvarez (A&M: Texas A&M University)H-Index: 5
Last. Peter P. Nghiem (A&M: Texas A&M University)H-Index: 9
view all 7 authors...
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRI...
4 CitationsSource
#1Petros Patsali (The Cyprus Institute of Neurology and Genetics)H-Index: 2
#2Marina Kleanthous (The Cyprus Institute of Neurology and Genetics)H-Index: 21
Last. Carsten W. Lederer (The Cyprus Institute of Neurology and Genetics)H-Index: 15
view all 3 authors...
Designer nucleases are versatile tools for genome modification and therapy development and have gained widespread accessibility with the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology. Prokaryotic RNA-guided nucleases of CRISPR/Cas type, since first being adopted as editing tools in eukaryotic cells, have experienced rapid uptake and development. Diverse modes of delivery by viral and non-viral vectors and ongoing discovery...
3 CitationsSource
#2Dmitry KostyushevH-Index: 3
Last. Vladimir ChulanovH-Index: 12
view all 6 authors...
The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small mol...
#1Margaret D. Weinroth (CSU: Colorado State University)H-Index: 5
#2Brianna C. Britton (CSU: Colorado State University)H-Index: 1
Last. Keith E. Belk (CSU: Colorado State University)H-Index: 46
view all 3 authors...
Abstract Detection, reduction, and public health monitoring of foodborne pathogens has advanced in precision and efficiency as technology has progressed. Here, we look back on the evolution of food safety management and public health, and attempt to provide a view of how the technology and tools have changed, and how emerging technologies and tools may impact how we manage food safety and public health in the future. With the revolution of gene editing techniques (e.g. CRISPR-Cas9, etc.), Next G...
view all 6 authors...
Advances in ex vivo technologies of human genome editing have made it possible to develop new approaches to the treatment of genetic, oncological, infectious and other diseases, which may involve the use of biomedical cell products. However, despite the rapid development of these technologies and a large number of clinical trials conducted in many countries around the world, only 4 products (Strimvelis, Zalmoxis, Kymriah and Yescarta) containing ex vivo genetically modified human cells are autho...
#1Jingwei Wei (AgResearch)H-Index: 4
#2Stefan Wagner (AgResearch)H-Index: 10
Last. Götz Laible (AgResearch)H-Index: 17
view all 10 authors...
We applied precise zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows’ milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the...
5 CitationsSource
#1Joyce C. Ohiri (NU: Northwestern University)H-Index: 2
With an increasing understanding of genetic defects leading to cardiomyopathy, focus is shifting to methods for correcting these underlying genetic defects. One approach involves treating mutant RNA through antisense oligonucleotides, and the first drug has now received regulatory approval to treat specific mutations associated with Duchenne muscular dystrophy. In contrast, gene editing targets DNA, and is currently being evaluated in the preclinical setting. For inherited cardiomyopathies, espe...
1 CitationsSource
#1Arildo Nerys-Junior (UFRJ: Federal University of Rio de Janeiro)H-Index: 2
#2Luciene P. Braga-Dias (FIOCRUZ: Oswaldo Cruz Foundation)H-Index: 2
Last. Amilcar Tanuri (UFRJ: Federal University of Rio de Janeiro)H-Index: 46
view all 5 authors...
: The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nuclea...
10 CitationsSource
#1John P. Budde (WashU: Washington University in St. Louis)H-Index: 22
#2Rita Martinez (WashU: Washington University in St. Louis)H-Index: 3
Last. Celeste M. Karch (WashU: Washington University in St. Louis)H-Index: 27
view all 9 authors...
Genome engineering in human induced pluripotent stem cells (iPSCs) represent an opportunity to examine the contribution of pathogenic and disease modifying alleles to molecular and cellular phenotypes. However, the practical application of genome-editing approaches in human iPSCs has been challenging. We have developed a precise and efficient genome-editing platform that relies on allele-specific guideRNAs (gRNAs) paired with a robust method for culturing and screening the modified iPSC clones. ...
3 CitationsSource
#1Wenyi Wu (CSU: Central South University)H-Index: 5
#2Luosheng Tang (CSU: Central South University)H-Index: 2
Last. Hetian Lei (MEE: Massachusetts Eye and Ear Infirmary)H-Index: 18
view all 4 authors...
Abstract The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease (Cas)9 is an effective instrument for revising the genome with great accuracy. This system has been widely employed to generate mutants in genomes from plants to human cells. Rapid improvements in Cas9 specificity in eukaryotic cells have opened great potential for the use of this technology as a therapeutic. Herein, we summarize the recent advancements of CRISPR-Cas9 use in r...
2 CitationsSource