190 Biallelic correction of recessive dystrophic epidermolysis bullosa mutations in iPSCs using CRISPR/Cas9- based genome editing

J. Jackow, Z. Guo, E. Abaci, Y. Doucet, C. Hansen, J. Salas-Alanis, A. Christiano

Research output: Contribution to journalMeeting Abstract

Abstract

Several approaches for correcting mutations have been reported using sequence-specificnucleases, which allow for efficient genetic modifications at targeted sites of interest. How-ever, the low efficiency drives up the cost and drug selection leads to safety concerns inclinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inheritedskin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7),which is the major constituent of anchoring fibrils at the basement membrane zone (BMZ).Patients with RDEB lack functional C7 and thus have severely impaired dermal-epidermalstability. Here, we successfully corrected a recurrent hotspot mutation in exon 19(c.2470insG) in theCOL7A1gene using homology-directed repair with CRISPR Cas9-gRNAin iPSCs derived from a patient with RDEB who was homozygous for this mutation. Weutilized single-strand oligodeoxynuleotides as the donor template, together with a high-fi-delity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic andmonoallelic correction ofCOL7A1mutations, which allowed us to select the targeted cells byFACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60mm2cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted inan efficiency of 10% for biallelic correction (WT/WT) and 40% for monoallelic correction(WT/mut). Moreover, Sanger sequence analysis indicated that the target sequence areas werefree of undesired mutations. These gene-corrected iPSCs can be differentiated into kerati-nocytes and fibroblasts forex vivo3D skin construction to assess the restoration of C7 proteinat the BMZ. Taken together, we have demonstrated correction of specific RDEB mutationswithout drug selection in a large locus such asCOL7A,which is a crucial step for clinicalapplications to develop innovative stem cell therapies for RDEB.
Original languageEnglish
Pages (from-to)S225
JournalJournal of Investigative Dermatology
Volume137
Issue number10
DOIs
Publication statusPublished - 1 Oct 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Epidermolysis Bullosa Dystrophica
Genes
Mutation
Collagen Type VII
Pharmaceutical Preparations
Basement Membrane
Fibroblasts
Stem cells
Restoration
Seed
Exons
Skin
Repair
Drug Costs
Cell- and Tissue-Based Therapy
Reporter Genes
Sequence Analysis
Seeds
Stem Cells

Cite this

Jackow, J. ; Guo, Z. ; Abaci, E. ; Doucet, Y. ; Hansen, C. ; Salas-Alanis, J. ; Christiano, A. / 190 Biallelic correction of recessive dystrophic epidermolysis bullosa mutations in iPSCs using CRISPR/Cas9- based genome editing. In: Journal of Investigative Dermatology. 2017 ; Vol. 137, No. 10. pp. S225.
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abstract = "Several approaches for correcting mutations have been reported using sequence-specificnucleases, which allow for efficient genetic modifications at targeted sites of interest. How-ever, the low efficiency drives up the cost and drug selection leads to safety concerns inclinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inheritedskin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7),which is the major constituent of anchoring fibrils at the basement membrane zone (BMZ).Patients with RDEB lack functional C7 and thus have severely impaired dermal-epidermalstability. Here, we successfully corrected a recurrent hotspot mutation in exon 19(c.2470insG) in theCOL7A1gene using homology-directed repair with CRISPR Cas9-gRNAin iPSCs derived from a patient with RDEB who was homozygous for this mutation. Weutilized single-strand oligodeoxynuleotides as the donor template, together with a high-fi-delity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic andmonoallelic correction ofCOL7A1mutations, which allowed us to select the targeted cells byFACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60mm2cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted inan efficiency of 10{\%} for biallelic correction (WT/WT) and 40{\%} for monoallelic correction(WT/mut). Moreover, Sanger sequence analysis indicated that the target sequence areas werefree of undesired mutations. These gene-corrected iPSCs can be differentiated into kerati-nocytes and fibroblasts forex vivo3D skin construction to assess the restoration of C7 proteinat the BMZ. Taken together, we have demonstrated correction of specific RDEB mutationswithout drug selection in a large locus such asCOL7A,which is a crucial step for clinicalapplications to develop innovative stem cell therapies for RDEB.",
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190 Biallelic correction of recessive dystrophic epidermolysis bullosa mutations in iPSCs using CRISPR/Cas9- based genome editing. / Jackow, J.; Guo, Z.; Abaci, E.; Doucet, Y.; Hansen, C.; Salas-Alanis, J.; Christiano, A.

In: Journal of Investigative Dermatology, Vol. 137, No. 10, 01.10.2017, p. S225.

Research output: Contribution to journalMeeting Abstract

TY - JOUR

T1 - 190 Biallelic correction of recessive dystrophic epidermolysis bullosa mutations in iPSCs using CRISPR/Cas9- based genome editing

AU - Jackow, J.

AU - Guo, Z.

AU - Abaci, E.

AU - Doucet, Y.

AU - Hansen, C.

AU - Salas-Alanis, J.

AU - Christiano, A.

PY - 2017/10/1

Y1 - 2017/10/1

N2 - Several approaches for correcting mutations have been reported using sequence-specificnucleases, which allow for efficient genetic modifications at targeted sites of interest. How-ever, the low efficiency drives up the cost and drug selection leads to safety concerns inclinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inheritedskin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7),which is the major constituent of anchoring fibrils at the basement membrane zone (BMZ).Patients with RDEB lack functional C7 and thus have severely impaired dermal-epidermalstability. Here, we successfully corrected a recurrent hotspot mutation in exon 19(c.2470insG) in theCOL7A1gene using homology-directed repair with CRISPR Cas9-gRNAin iPSCs derived from a patient with RDEB who was homozygous for this mutation. Weutilized single-strand oligodeoxynuleotides as the donor template, together with a high-fi-delity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic andmonoallelic correction ofCOL7A1mutations, which allowed us to select the targeted cells byFACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60mm2cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted inan efficiency of 10% for biallelic correction (WT/WT) and 40% for monoallelic correction(WT/mut). Moreover, Sanger sequence analysis indicated that the target sequence areas werefree of undesired mutations. These gene-corrected iPSCs can be differentiated into kerati-nocytes and fibroblasts forex vivo3D skin construction to assess the restoration of C7 proteinat the BMZ. Taken together, we have demonstrated correction of specific RDEB mutationswithout drug selection in a large locus such asCOL7A,which is a crucial step for clinicalapplications to develop innovative stem cell therapies for RDEB.

AB - Several approaches for correcting mutations have been reported using sequence-specificnucleases, which allow for efficient genetic modifications at targeted sites of interest. How-ever, the low efficiency drives up the cost and drug selection leads to safety concerns inclinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inheritedskin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7),which is the major constituent of anchoring fibrils at the basement membrane zone (BMZ).Patients with RDEB lack functional C7 and thus have severely impaired dermal-epidermalstability. Here, we successfully corrected a recurrent hotspot mutation in exon 19(c.2470insG) in theCOL7A1gene using homology-directed repair with CRISPR Cas9-gRNAin iPSCs derived from a patient with RDEB who was homozygous for this mutation. Weutilized single-strand oligodeoxynuleotides as the donor template, together with a high-fi-delity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic andmonoallelic correction ofCOL7A1mutations, which allowed us to select the targeted cells byFACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60mm2cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted inan efficiency of 10% for biallelic correction (WT/WT) and 40% for monoallelic correction(WT/mut). Moreover, Sanger sequence analysis indicated that the target sequence areas werefree of undesired mutations. These gene-corrected iPSCs can be differentiated into kerati-nocytes and fibroblasts forex vivo3D skin construction to assess the restoration of C7 proteinat the BMZ. Taken together, we have demonstrated correction of specific RDEB mutationswithout drug selection in a large locus such asCOL7A,which is a crucial step for clinicalapplications to develop innovative stem cell therapies for RDEB.

U2 - 10.1016/j.jid.2017.07.187

DO - 10.1016/j.jid.2017.07.187

M3 - Meeting Abstract

VL - 137

SP - S225

JO - Journal of Investigative Dermatology

JF - Journal of Investigative Dermatology

SN - 0022-202X

IS - 10

ER -