CRISPR-Cas9-Mediated Correction of the 1.02 kb Common Deletion in CLN3 in Induced Pluripotent Stem Cells from Patients with Batten Disease

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 induced pluripotent stem cells generated from two independent patients: one homozygous and one compound heterozygous for the 1.02 kb deletion. Our strategy included delivery of a construct that carried >3 kb of DNA: wild-type CLN3 sequence and a LoxP-flanked, puromycin resistance cassette for positive selection. This strategy resulted in correction at the genomic DNA and mRNA levels in the two independent patient lines. These CRISPR-corrected isogenic cell lines will be a valuable tool for disease modeling and autologous retinal cell replacement.

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9

“…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].…”

“…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.…”

“…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].…”

“…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).…”

“…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.…”