Abstract:Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a rare progressive neurodegenerative disorder caused by mutations in CLN3. Patients present with early-onset retinal degeneration, followed by epilepsy, progressive motor deficits, cognitive decline, and premature death. Approximately 85% of individuals with Batten disease harbor at least one allele containing a 1.02 kb genomic deletion spanning exons 7 and 8. This study demonstrates CRISPR-Cas9-based homology-dependent repair of this mutation in indu… Show more
“…As indicated above, to prevent re-cleavage events, we included a synonymous variant in the sg4 PAM site (CGG>CGA). To select for cells that incorporated the HDR sequence, a puromycin resistance cassette under the control of the mPGK promoter was added to intron 1 as previously described [16,18]. In addition, the stop codon in the puromycin resistance sequence was replaced with a porcine 2A peptide (P2A) coding sequence, followed by the Herpes Simplex Virus type 1 thymidine kinase (vTK) gene (thymidine kinase phosphorylates ganciclovir, a nucleoside analog, which disrupts DNA synthesis and induces cell death) and a downstream polyadenylation sequence (PA) [16].…”
Section: Resultsmentioning
confidence: 99%
“…The sgRNAs used in this study were designed to target the region of NR2E3 that contains the c.119-2A>C (Patient 1) and c.219G>C; p.(Arg73Ser) (Patient 2) mutations using the Benchling platform () or the Optimized CRISPR Design Tool (crispr.mit.edu). Guides were cloned into our previously described bicistronic construct expressing a human codon-optimized Streptococcus pyogenes Cas9 ( spCas9 ) nuclease [17,18]. A homology-directed repair (HDR) construct was synthesized by GenScript.…”
Section: Methodsmentioning
confidence: 99%
“…sgRNA and CRISPR-Cas9 constructs were transfected into HEK293T cells (ATCC) using Lipofectamine 2000 (Thermo Fisher Scientific) and cleavage was assessed via a T7 endonuclease 1 (T7E1) assay using NR2E3 -specific primers (Table S1) as described previously [17,18]. Control iPSCs were transfected with 2 μg of plasmid using the Neon transfection system as described previously [16].…”
Section: Methodsmentioning
confidence: 99%
“…The CRISPR-Cas9 machinery was delivered to iPSCs and analyzed as described previously [16,17,18]. Briefly, the sg4-spCas9 and HDR plasmids were transfected at a 1:2 molar ratio into patient iPSCs with Lipofectamine Stem (Thermo Fisher Scientific; Patient 1) or Neon electroporation (Thermo Fisher Scientific; Patient 2).…”
Section: Methodsmentioning
confidence: 99%
“…To correct retinal disease-causing variants in patient-derived iPSCs, we and others have used CRISPR-based genome editing [15,16,17,18,19,20,21,22]. Unlike the more cumbersome ZFN- and TALEN-based approaches, which require the development of elaborate genomic targeting complexes, the CRISPR method relies on the use of small single guide RNAs (sgRNAs) that can be easily synthesized to direct human codon-optimized Cas9 nuclease to specific genomic targets.…”
Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.
“…As indicated above, to prevent re-cleavage events, we included a synonymous variant in the sg4 PAM site (CGG>CGA). To select for cells that incorporated the HDR sequence, a puromycin resistance cassette under the control of the mPGK promoter was added to intron 1 as previously described [16,18]. In addition, the stop codon in the puromycin resistance sequence was replaced with a porcine 2A peptide (P2A) coding sequence, followed by the Herpes Simplex Virus type 1 thymidine kinase (vTK) gene (thymidine kinase phosphorylates ganciclovir, a nucleoside analog, which disrupts DNA synthesis and induces cell death) and a downstream polyadenylation sequence (PA) [16].…”
Section: Resultsmentioning
confidence: 99%
“…The sgRNAs used in this study were designed to target the region of NR2E3 that contains the c.119-2A>C (Patient 1) and c.219G>C; p.(Arg73Ser) (Patient 2) mutations using the Benchling platform () or the Optimized CRISPR Design Tool (crispr.mit.edu). Guides were cloned into our previously described bicistronic construct expressing a human codon-optimized Streptococcus pyogenes Cas9 ( spCas9 ) nuclease [17,18]. A homology-directed repair (HDR) construct was synthesized by GenScript.…”
Section: Methodsmentioning
confidence: 99%
“…sgRNA and CRISPR-Cas9 constructs were transfected into HEK293T cells (ATCC) using Lipofectamine 2000 (Thermo Fisher Scientific) and cleavage was assessed via a T7 endonuclease 1 (T7E1) assay using NR2E3 -specific primers (Table S1) as described previously [17,18]. Control iPSCs were transfected with 2 μg of plasmid using the Neon transfection system as described previously [16].…”
Section: Methodsmentioning
confidence: 99%
“…The CRISPR-Cas9 machinery was delivered to iPSCs and analyzed as described previously [16,17,18]. Briefly, the sg4-spCas9 and HDR plasmids were transfected at a 1:2 molar ratio into patient iPSCs with Lipofectamine Stem (Thermo Fisher Scientific; Patient 1) or Neon electroporation (Thermo Fisher Scientific; Patient 2).…”
Section: Methodsmentioning
confidence: 99%
“…To correct retinal disease-causing variants in patient-derived iPSCs, we and others have used CRISPR-based genome editing [15,16,17,18,19,20,21,22]. Unlike the more cumbersome ZFN- and TALEN-based approaches, which require the development of elaborate genomic targeting complexes, the CRISPR method relies on the use of small single guide RNAs (sgRNAs) that can be easily synthesized to direct human codon-optimized Cas9 nuclease to specific genomic targets.…”
Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.
Neurodegenerative diseases are prominent causes of pain, suffering, and death worldwide. Traditional approaches modelling neurodegenerative diseases are deficient, and therefore, improved strategies that effectively recapitulate the pathophysiological conditions of neurodegenerative diseases are the need of the hour. The generation of human-induced pluripotent stem cells (iPSCs) has transformed our ability to model neurodegenerative diseases in vitro and provide an unlimited source of cells (including desired neuronal cell types) for cell replacement therapy. Recently, CRISPR/Cas9-based genome editing has also been gaining popularity because of the flexibility they provide to generate and ablate disease phenotypes. In addition, the recent advancements in CRISPR/Cas9 technology enables researchers to seamlessly target and introduce precise modifications in the genomic DNA of different human cell lines, including iPSCs. CRISPR-iPSC-based disease modelling, therefore, allows scientists to recapitulate the pathological aspects of most neurodegenerative processes and investigate the role of pathological gene variants in healthy non-patient cell lines. This review outlines how iPSCs, CRISPR/Cas9, and CRISPR-iPSC-based approaches accelerate research on neurodegenerative diseases and take us closer to a cure for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis, and so forth.
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