Journal: Nature Communications

Article Title: CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation

doi: 10.1038/s41467-019-12028-5

Figure Lengend Snippet: ATI and pseudo-mRNAs contribute to foreign protein production in CRISPR-edited cell line. a Genomic structure of the LKB1 gene and the exonic sequence targeted by the LKB1 exon 1 sgRNA. b Emergence of a small LKB1 protein (ATI LKB1) as a consequence of CRISPR-Cas9 gene editing. Lysates generated from CRISPR-edited HAP1 clones were subjected to western blot analysis using two distinct LBK1 antibodies recognizing either N- and C-terminus localized epitopes. c Western blot analysis of CRISPR-Cas9-edited MIA clones reveals the appearance of a large LKB1 protein (Super LKB1) in addition to the ATI LKB1 protein. d Genomic sequences of CRISPR-Cas9-edited HAP1 and MIA clones reveal on-target insertion/deletion mutations in the LKB1 gene. Predicted gene alteration for each clone is indicated. e CRISPR-Cas9-introduced INDELs are associated with the expression of an LKB1 pseudo-mRNA transcript. RT-PCR analysis was performed using primers mapping to 5′ UTR and exon 4 in LKB1 to generate amplicons from the cDNA of CRISPR-Cas9-edited clones. MIA clones M2 and M3, which express Super LKB1 protein, harbor an mRNA species that includes an additional exon. The 131 bp additional exon contains canonical splice acceptor and donor sequences. f A cDNA expression strategy for understanding allele-specific CRISPR-introduced INDELs on protein expression provides evidence for ATI. LKB1 and Super LKB1 cDNA expression constructs harboring genomic alterations found in LKB1 of MIA Clone M2 were introduced into HELA cells that lack endogenous LKB1 expression. The 1 bp insertion or 2 bp deletion in the Super LKB1 cDNA result in proteins that co-migrate with the Super LKB1 protein observed in MIA Clone M2. On the other hand, the same mutations in LKB1 cDNA give rise to proteins that co-migrate with the ATI LKB1 protein found in Clone M2, and with the protein that initiates at Met51. Source data are provided as a Source Data file

Article Snippet: RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed on 1 μg of RNA using the ProtoScript First Strand cDNA Synthesis Kit (Promega).

Techniques: CRISPR, Sequencing, Generated, Clone Assay, Western Blot, Genomic Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct

Journal: Nature Communications

Article Title: CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation

doi: 10.1038/s41467-019-12028-5

Figure Lengend Snippet: Compromised ESEs account for INDEL-induced exon skipping. a Genomic structure of SUFU and exonic sequence targeted by SUFU exon 8 sgRNA. The recognition sites of antibodies used in panel b are indicated. b Western blot analysis of HAP1 cells edited with SUFU exon 8 sgRNA shows no detectable expression of SUFU. c Exon skipping is prevalent in CRISPR-Cas9-edited SUFU clones. RT-PCR analysis using primers flanking exons 6 and 10 of SUFU in CRISPR-Cas9-edited SUFU clones. Sequencing of amplicons reveals exon skipping in all of clones except clones H9 and H10. d Disruption of exon splicing enhancers (ESEs) by CRISPR-introduced INDELs triggers skipping of the edited exons. Genetic mutation and the presence/absence of exon skipping events for each clone are indicated. Putative ESEs were identified using the RESCUE-ESE web server. e Multiple sgRNA sequences located in symmetric or asymmetric exons of the SUFU gene used for targeted disruption of ESEs. f sgRNAs described in “ e ” were used to edit the SUFU gene in RMS13 cells. Western blot analysis of lysates derived from the CRISPR-Cas9-edited RMS13 clones show no detectable SUFU protein. g Genomic sequences of RMS13 clones edited with SUFU exon 3 sgRNAs. CRISPR-introduced mutations and putative exon splicing enhancer (ESE) and exon splicing silencer (ESS) sequences are indicated. h RT-PCR analysis and cDNA sequencing result of clones R2 and R3 using primers flanking exon 1 and 5. i Genomic sequences of RMS13 clones edited with SUFU exon 2 sgRNA. CRISPR-introduced mutations and putative ESE/ESS sequences are indicated. j RT-PCR analysis and cDNA sequencing result of clones R1 and R4 using primers flanking 5′ UTR and exon 4. k Genomic sequences of RMS13 clones edited with SUFU exon 8 sgRNA. CRISPR-introduced mutations and putative ESE and ESS sequences are indicated. l RT-PCR analysis and cDNA sequencing result of the clones R1 and R4 using primers flanking exon 6 and exon 10. m Disruption of ESE code is highly reliable in anticipating CRISPR-Cas9-induced exon skipping. Twenty-four CRISPR-Cas9-edited cell lines with different mutations were analyzed for the presence/absence of exon skipping events and changes in ESE sequences due to CRISPR-introduced INDELs. Source data are provided as a Source Data file

Article Snippet: RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed on 1 μg of RNA using the ProtoScript First Strand cDNA Synthesis Kit (Promega).

Techniques: Sequencing, Western Blot, Expressing, CRISPR, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Disruption, Mutagenesis, Derivative Assay, Genomic Sequencing

Journal: Nature Communications

Article Title: CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation

doi: 10.1038/s41467-019-12028-5

Figure Lengend Snippet: Targeting RNA-regulatory elements for gene knockout agendas. a CRISPinatoR: a web-based guide RNA design tool that utilizes targeted ESE disruption for achieving gene elimination. CRISPinatoR identifies sgRNA sequences that target ESEs in asymmetric exons and calculates off-targeting potential and the number of splice variants impacted by the sgRNAs. A scoring system that integrates all three parameters is used to provide sgRNAs with high gene knockout potential. b Genome structure of the LRP5 gene and sgRNA sequences targeting the asymmetric exon 2 and the symmetric exon 16. c Genomic sequencing results of HAP1 clones edited using LRP5 exon 2 and exon 16 sgRNAs. CRISPR-introduced mutations and the putative ESE sequences are indicated. d Exclusion of an asymmetric or a symmetric exon with INDEL-induced changes to the putative ESE sequences. RT-PCR analysis and cDNA sequencing result of HAP1 cells edited with LRP5 exon 2 and exon 16 sgRNAs. e Targeted ESE disruption in asymmetric exon increases gene knockout potential. Western blot analysis of HAP1 clones edited with LRP5 exon 2 sgRNA (Clone 21) and exon 16 sgRNA (Clone 3) was probed with two distinct antibodies indicated in “ b ”. ESE disruption in symmetric exon 2 produces internally truncated in-frame LRP5 protein. f The internally truncated LRP5 protein is glycosylated. Lysates derived from WT or LRP5 ΔE16 HAP1 cells were incubated with the deglycosidase PNGase F then subjected to western blot analysis. g Exclusion of LRP5 exon16 would delete a sequence adjacent to the WNT3A binding domains. h The LRP5 ΔE16 protein formed post skipping of a symmetric exon is functionally active. WNT/β-catenin pathway activity in response to WNT3A conditioned medium (WNT3A CM) was measured for HAP1 WT, LRP5 ΔE2, and LRP5 ΔE16 cells. WNT pathway inhibitors WNT974 (PORCNi) and IWR1 (TNKSi) serve as negative and positive control, respectively. All error bars represent mean of triplicates ± s.d. The experiment was repeated three times with similar results. Statistical testing was performed using Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file

Article Snippet: RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed on 1 μg of RNA using the ProtoScript First Strand cDNA Synthesis Kit (Promega).

Techniques: Gene Knockout, Disruption, Genomic Sequencing, Clone Assay, CRISPR, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Derivative Assay, Incubation, Binding Assay, Activity Assay, Positive Control