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Genome engineering of isogenic human ES cells to model autism disorders.

Martinez RA, Stein JL, Krostag AR, Nelson AM, Marken JS, Menon V, May RC, Yao Z, Kaykas A, Geschwind DH, Grimley JS - Nucleic Acids Res. (2015)

Bottom Line: Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background.We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay.We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.

View Article: PubMed Central - PubMed

Affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.

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Related in: MedlinePlus

Description of TALENSeek, an automated TALEN-binding site detection algorithm. (a) Automated annotation of genomic regions is used to identify start and end sites of a gene of interest using the farthest protein coding regions across all isoforms (a super-isoform). (b) Potential TALEN-binding sites are identified. (c) The sites are evaluated for uniqueness across the genome. (d) If a binding site is not unique, the search continues further into the gene and the algorithm iterates from step (b). (e) The output .bed file allows visualization of the TALEN-binding sites in the UCSC genome browser, shown with human genome build GRCh37 (hg19).
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Figure 1: Description of TALENSeek, an automated TALEN-binding site detection algorithm. (a) Automated annotation of genomic regions is used to identify start and end sites of a gene of interest using the farthest protein coding regions across all isoforms (a super-isoform). (b) Potential TALEN-binding sites are identified. (c) The sites are evaluated for uniqueness across the genome. (d) If a binding site is not unique, the search continues further into the gene and the algorithm iterates from step (b). (e) The output .bed file allows visualization of the TALEN-binding sites in the UCSC genome browser, shown with human genome build GRCh37 (hg19).

Mentions: TALENSeek first creates the super-isoform, the furthest 5′ and 3′ length across all isoforms defined in ENSEMBL, for the specified gene (Figure 1a). Near the transcriptional start and stop sites, an acceptable TALEN-binding site pair is sought where the pairs are separated by 15–30 bp and each 14–19 bp binding site begins with a T. The program searches iteratively into the gene until an acceptable site is found (Figure 1b and c). This site is then searched for uniqueness in the genome, allowing for a number of user-defined mismatches. If a homologous sequence in human and mouse is specified, the binding site identified in the human genome is tested to be within 250 bp of the start or end site of that same gene in the mouse genome (Figure 1d). The user has the option to test if the TALEN-binding site is unique in each genome. The output includes a .bed file for visualization in the UCSC genome browser (38) that identifies the individual TALEN-binding sites in the human and mouse genomes for each specified target (Figure 1e).


Genome engineering of isogenic human ES cells to model autism disorders.

Martinez RA, Stein JL, Krostag AR, Nelson AM, Marken JS, Menon V, May RC, Yao Z, Kaykas A, Geschwind DH, Grimley JS - Nucleic Acids Res. (2015)

Description of TALENSeek, an automated TALEN-binding site detection algorithm. (a) Automated annotation of genomic regions is used to identify start and end sites of a gene of interest using the farthest protein coding regions across all isoforms (a super-isoform). (b) Potential TALEN-binding sites are identified. (c) The sites are evaluated for uniqueness across the genome. (d) If a binding site is not unique, the search continues further into the gene and the algorithm iterates from step (b). (e) The output .bed file allows visualization of the TALEN-binding sites in the UCSC genome browser, shown with human genome build GRCh37 (hg19).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446412&req=5

Figure 1: Description of TALENSeek, an automated TALEN-binding site detection algorithm. (a) Automated annotation of genomic regions is used to identify start and end sites of a gene of interest using the farthest protein coding regions across all isoforms (a super-isoform). (b) Potential TALEN-binding sites are identified. (c) The sites are evaluated for uniqueness across the genome. (d) If a binding site is not unique, the search continues further into the gene and the algorithm iterates from step (b). (e) The output .bed file allows visualization of the TALEN-binding sites in the UCSC genome browser, shown with human genome build GRCh37 (hg19).
Mentions: TALENSeek first creates the super-isoform, the furthest 5′ and 3′ length across all isoforms defined in ENSEMBL, for the specified gene (Figure 1a). Near the transcriptional start and stop sites, an acceptable TALEN-binding site pair is sought where the pairs are separated by 15–30 bp and each 14–19 bp binding site begins with a T. The program searches iteratively into the gene until an acceptable site is found (Figure 1b and c). This site is then searched for uniqueness in the genome, allowing for a number of user-defined mismatches. If a homologous sequence in human and mouse is specified, the binding site identified in the human genome is tested to be within 250 bp of the start or end site of that same gene in the mouse genome (Figure 1d). The user has the option to test if the TALEN-binding site is unique in each genome. The output includes a .bed file for visualization in the UCSC genome browser (38) that identifies the individual TALEN-binding sites in the human and mouse genomes for each specified target (Figure 1e).

Bottom Line: Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background.We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay.We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.

View Article: PubMed Central - PubMed

Affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.

Show MeSH
Related in: MedlinePlus