CRISPR Del/Rei: a simple, flexible, and efficient pipeline for scarless genome editing

Feuer, Kyra L; Wahbeh, Marah H.; Yovo, Christian; Rabie, Eman; Lam, Anh-Thu N.; Abdollahi, Sara; Young, Lindsay J.; Rike, Bailey; Umamageswaran, Akul; Avramopoulos, Dimitrios

doi:10.1038/s41598-022-16004-w

Cited by 3 publications

(16 citation statements)

References 17 publications

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“…We received the line at passage number 12. The iPSC line was treated as we have outlined previously 21, 29 . Briefly, the cells were grown on culture plates coated with 5μg/mL laminin (ThermoFisher BioLamina #LN521) and incubated in a humidified 5% CO 2 incubator at 37°C for ≥2 hours.…”

Section: Methodsmentioning

confidence: 99%

“…Cell stocks were first frozen at −80°C in StemFlex media with 10% Dimethylsulfoxide (DMSO) for 24h before storage in liquid nitrogen. iPSC genotyping was done by PCR and Sanger sequencing using primers listed in the Supplementary Information file of Feuer & Wabeh, 2022 (see Online Methods; TSNARE1 experiment) 21 .…”

Section: Methodsmentioning

confidence: 99%

“…To induce target sequence deletion and subsequent re-insertion of the rs4129585 non-risk (A) and risk (C) alleles, we employed our pipeline for scarless genome editing, CRISPR Del/Rei 21 . Detailed methods, transfection and selection details, and summary of off-target analyses; as well as sequences for primers, single guide RNAs (sgRNAs), and single stranded oligodeoxynucleotide (ssODN), can be found in the Supplementary Information file of Feuer & Wahbeh et al ., 2022 (see Online Methods; TSNARE1 experiment) 21 . In brief, sgRNA oligos were designed using CRISPOR (http://crispor.tefor.net/crispor.py) to flank rs4129585 and induce a deletion between GRCh37/hg19 chr8:142231553-142231597.…”

Section: Methodsmentioning

confidence: 99%

“…Restriction enzyme BbsI sticky ends were added to single-stranded DNA oligos before the oligos were annealed and cloned in pairs into pDG459 plasmids containing a puromycin resistance gene. pDG459 plasmids were transfected into iPSCs using lipofectamine-stem and successful transfection was selected for using puromycin 21 . Cells were screened for desired edits and candidate off-target effects 21 at 4 genomic locations that showed 3 or fewer mismatches with the gRNAs and were in coding sequences or overlapped with DNAse hypersensitivity sites from the encode project (www.encodeproject.org; file hg38_wgEncodeRegDnaseClustered.txt).…”

Section: Methodsmentioning

confidence: 99%

“…Understanding how SCZ associated variants are involved in regulatory activities will promote the generation of innovative disease treatments and prophylactics. Technological advancements in cell engineering and genome editing using CRISPR/Cas9 21 – and the ability to generate human induced pluripotent stem cells (hiPSCs) that can be differentiated to a variety of cell types – have created a new toolkit for modeling human diseases 22 . Neurons that carry variants implicated in disease risk can now be generated and compared to neurons that carry non-risk alleles at the same position, on an otherwise isogenic background.…”

Section: Introductionmentioning

confidence: 99%

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A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

Wahbeh,

Boyd,

Yovo

et al. 2023

Preprint

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Recent collaborative genome wide association studies (GWAS) have identified >200 independent loci contributing to risk for schizophrenia (SCZ). The genes closest to these loci have diverse functions, supporting the potential involvement of multiple relevant biological processes; yet there is no direct evidence that individual variants are functional or directly linked to specific genes. Nevertheless, overlap with certain epigenetic marks suggest that most GWAS-implicated variants are regulatory. Based on the strength of association with SCZ and the presence of regulatory epigenetic marks, we chose one such variant nearTSNARE1andADGRB1, rs4129585, to test for functional potential and assay differences that may drive the pathogenicity of the risk allele. We observed that the variant-containing sequence drives reporter expression in relevant neuronal populations in zebrafish. Next, we introduced each allele into human induced pluripotent cells and differentiated 4 isogenic clones homozygous for the risk allele and 5 clones homozygous for the non-risk allele into neural precursor cells. Employing RNA-seq, we found that the two alleles yield significant transcriptional differences in the expression of 109 genes at FDR <0.05 and 259 genes at FDR <0.1. We demonstrate that these genes are highly interconnected in pathways enriched for synaptic proteins, axon guidance, and regulation of synapse assembly. Exploration of genes near rs4129585 suggests that this variant does not regulateTSNARE1transcripts, as previously thought, but may regulate the neighboringADGRB1, a regulator of synaptogenesis. Our results suggest that rs4129585 is a functional common variant that functions in specific pathways likely involved in SCZ risk.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

Wahbeh,

Boyd,

Yovo

et al. 2023

Preprint

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A functional schizophrenia-associated genetic variant near the TSNARE1 and ADGRB1 genes

Wahbeh,

Boyd,

Yovo

et al. 2024

Human Genetics and Genomics Advances

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A Missense Variant in CASKIN1’s Proline-Rich Region Segregates with Psychosis in a Three-Generation Family

Wahbeh

Peng²,

Bacharaki³

et al. 2023

Genes

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The polygenic nature of schizophrenia (SCZ) implicates many variants in disease development. Rare variants of high penetrance have been shown to contribute to the disease prevalence. Whole-exome sequencing of a large three-generation family with SCZ and bipolar disorder identified a single segregating novel, rare, non-synonymous variant in the gene CASKIN1. The variant D1204N is absent from all databases, and CASKIN1 has a gnomAD missense score Z = 1.79 and pLI = 1, indicating its strong intolerance to variation. We find that introducing variants in the proline-rich region where the D1204N resides results in significant cellular changes in iPSC-derived neurons, consistent with CASKIN1’s known functions. We observe significant transcriptomic changes in 368 genes (padj < 0.05) involved in neuronal differentiation and nervous system development. We also observed nominally significant changes in the frequency of action potentials during differentiation, where the speed at which the edited and unedited cells reach the same level of activity differs. Our results suggest that CASKIN1 is an excellent gene candidate for psychosis development with high penetrance in this family.

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CRISPR Del/Rei: a simple, flexible, and efficient pipeline for scarless genome editing

Cited by 3 publications

References 17 publications

A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

A functional schizophrenia-associated genetic variant near the TSNARE1 and ADGRB1 genes

A Missense Variant in CASKIN1’s Proline-Rich Region Segregates with Psychosis in a Three-Generation Family

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