Journal: Circulation Research

Article Title: RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function

doi: 10.1161/CIRCRESAHA.122.321728

Figure Lengend Snippet: Human RBPMS2 - (RNA-binding protein with multiple splicing [variants] 2)-null cardiomyocytes exhibit myofibrillar disarray and calcium handling defects similar to those in zebrafish rbpms2 -null cardiomyocytes. A , Schematic diagrams of human RBPMS2 ( top ) and the predicted protein product of the Δ RBPMS2 null allele created with CRISPR/Cas9-mediated genome editing ( bottom ). The asterisk shows the location of a premature stop codon within the RRM (RNA-recognition motifs) caused by a frame-shifting 2 base pair deletion. The white box shows the location of divergent amino acids before the stop codon. B and C , Bar graphs showing the relative expression levels of RBPMS2 in wild-type control (CTRL) and ΔRBPMS2 human induced pluripotent stem cells (hiPSCs; B ) and cardiomyocytes (hiPSC-CMs; C ) after 15 days of directed differentiation. n=6 biological replicates/group, each with three technical replicates. Error bars show one standard deviation. Statistical significance was determined by an unpaired, 2-tailed Student's t test assuming equal variances. Significant P values are shown. D–K , Representative confocal projections of CTRL ( D–G ) and ΔRBPMS2 ( H–K ) hiPSC-CMs immunostained with antibodies that detect cardiac troponin T (CT3; green) or alpha-actinin (anti-sarcomeric alpha-actinin; magenta) to visualize thin filaments or Z-disks, respectively, and counterstained with DAPI (4'‚6-diamidino-2-phenylindole; blue). Single ( D–F; H–J ) and merged triple ( G and K ) channel images are shown. n=50 cells/group from at least 3 wells from 2 separate differentiations. L and M , Representative traces of fluorescence intensity over baseline (F/F o ; top ) derived from line scans ( bottom ) of CTRL ( L ) and ΔRBPMS2 ( M ) hiPSC-CMs loaded with fluo-4 and paced at 1 Hz. N–S , Violin plots showing calcium transient amplitude ( N ), duration at 80% (CaTD80; O ) and 50% (CaTD50; P ) repolarization, time-to-peak amplitude ( Q ), upstroke velocity ( R ), and unpaced spontaneous beating rate ( S ). n=80 cells/group, 40 each from 2 separate differentiations. Statistical significance was determined by an unpaired, 2-tailed Student's t test assuming equal variances or a Mann-Whitney U test. Significant P values are shown. Scale bars=25 μm.

Article Snippet: We previously described methods to generate a doxycycline-inducible Cas9 line in the WTC-11 iPSC background (Coriell Institute).

Techniques: RNA Binding Assay, CRISPR, Expressing, Control, Standard Deviation, Fluorescence, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

doi: 10.1101/2022.05.02.490368

Figure Lengend Snippet: GuideScan2 enables the design of gRNAs or gRNA libraries while exactly accounting for off-target effects. GuideScan CRIPSR-Cas9 and -Cas12a (Cpf1) libraries are precomputed for standard genomes; databases for custom genomes can be constructed with the command line tool. GuideScan2 builds a memory-efficient compressed index based on Burrows-Wheeler Transform to annotate all sufficiently specific gRNAs, enabling multiple design and analysis tasks.

Article Snippet: A549 cells expanded from a single clone harboring a doxycycline-inducible Cas9 (Addgene #114010) were a gift from J.T.

Techniques: Construct

Journal: Nature Communications

Article Title: Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

doi: 10.1038/s41467-022-28137-7

Figure Lengend Snippet: a gRNA (hybridized crRNA:tracrRNA) from S. pyogenes for a canonically folded gRNA; and b a predicted non-canonical folded gRNA ( SSH2 ). The spacer sequence, as well as the structural motifs nexus, 1st hairpin, and 2nd hairpin are shown. c Locked tracrRNA (t-lock) gRNA design carrying an elongated 1st hairpin with a superstable loop that should serve as a gRNA folding nucleation site and prevent misfolding. d Genome editing efficiencies for t0 (normal), t-lock (red), and commercial t0 (IDT) (black frame). t0 (IDT) has the same nucleotide sequence as t0, but carries additional proprietary chemical modifications. Independent biological replicates are depicted by black dots ( n = 3, except PIGZ t0 n = 2) and the error bars show the SEM. The panels show the efficiencies for 10 different gRNAs with different predicted non-canonical spacer interactions to the nexus (cyan), 1st hairpin (pink), 2nd hairpin (blue), and/or the spacer sequence itself (orange). The predicted crRNA:tracrRNA structure from the RNA structure web server is shown below the spacer sequence. The folding predictions with the locked tracrRNA are identical except for an elongated 1st hairpin and the formation of a 1st hairpin in the case of OSBP2 and C3 . e Box plots of relative genome editing efficiencies for gRNAs from d ( n = 10) with respect to the corresponding t0 tracrRNA set to 100%. Boxes extend from the 25th to 75th percentile and show the median as a line. Whiskers extend from the minimum to the maximum values. Chemically synthesized gRNAs (hybridized crRNA:tracrRNA) were lipofected into Cas9 expressing 409-B2 iCRISPR human induced pluripotent stem cells (hiPSCs). Source data are provided as a Source Data file.

Article Snippet: We used a human induced pluripotent stem cell (hiPSC) line (409-B2) carrying a doxycycline-inducible Cas9 (iCRISPR-Cas9) gene to test the efficiency of normal tracrRNA (t0), the locked tracrRNA (t-lock), and the normal tracrRNA with proprietary chemical modifications (t0-IDT) from a commercial provider in combination with ten different crRNAs, most of which have predicted strong non-canonical interactions to different regions in the gRNA (Fig. ).

Techniques: Sequencing, Synthesized, Expressing

Journal: Nature Communications

Article Title: Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

doi: 10.1038/s41467-022-28137-7

Figure Lengend Snippet: a Nucleotides of the gRNA (hybridized crRNA:tracrRNA) of which the 2’OH interacts with Cas9 or with residues of the nexus (cyan) are underlaid in red or circled in dark red, respectively. The residue underlaid in rose interacts with both Arg75 and Tyr52 of the bridge helix that modulates DNA cleavage. b Close-up of the crystal structure of the nexus and interacting residues adapted from NDB: 5F9R. Polar interactions are shown as dotted lines and those of 2′OH residues are shown as dotted red lines. c Single RNA nucleotide structure and chemical modifications. d Chemical modifications of the tracrRNA with phosphorothioate bonds and 2’OMe modifications (t-2′OMe*). The nexus loop of t-2′OMe-2.0* only contains unmodified RNA nucleotides (colored according to c). t0* only contains phosphorothioate bonds. e Genome editing efficiency of different chemically modified tracrRNAs hybridized with a medium cleavage crRNA electroporated into Cas9 expressing 409-B2 iCRISPR (hiPSCs). f Structure and chemical modifications of the genome editing optimized locked design (GOLD) tracrRNA. g Spacer sequences of previously described inactive gRNAs. Putative cleavage-inhibiting motifs are highlighted orange. h Genome editing efficiency of normal tracrRNA (blue), t-lock tracrRNA (red), and GOLD tracrRNA (gold) hybridized with crRNAs from g after electroporation into Cas9 expressing 409-B2 iCRISPR hiPSCs. Independent biological replicates ( n = 2) are depicted by black dots and the error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: We used a human induced pluripotent stem cell (hiPSC) line (409-B2) carrying a doxycycline-inducible Cas9 (iCRISPR-Cas9) gene to test the efficiency of normal tracrRNA (t0), the locked tracrRNA (t-lock), and the normal tracrRNA with proprietary chemical modifications (t0-IDT) from a commercial provider in combination with ten different crRNAs, most of which have predicted strong non-canonical interactions to different regions in the gRNA (Fig. ).

Techniques: Modification, Expressing, Electroporation

Journal: Nature Communications

Article Title: Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage

doi: 10.1038/s41467-022-28137-7

Figure Lengend Snippet: a Sketches of theoretical ‘worst-case’ spacers where the spacer is perfectly complementary to the 3′ end of the tracrRNA (roman numeral I), forms the locked hairpin with itself (roman numeral II), is perfectly complementary to the 3′ end of the crRNA (roman numeral III), or forms a perfect hairpin along the whole spacer sequence (roman numeral IV). The complementary region is colored orange and expected interactions are presented as hairpins or indicated by red arrows. b Strategy to introduce the theoretical ‘worst-case’ spacer target sequences from c into 409-B2 iCRISPR hiPSCs. Cleavage of the FRMD7 locus by Cas9 HiFi RNP is followed by subsequent HDR using a ssODN donor sequence carrying the intended target site next to a TGG PAM. M3814 is added to block NHEJ and increase HDR efficiency. Four different edits are done for target insertions I–IV followed by generation of single cell-derived cellular clones. c Genome editing efficiency of theoretical ‘worst-case’ gRNAs from a and t0 or GOLD tracrRNA. gRNAs were electroporated into Cas9 expressing 409-B2 iCRISPR hiPSCs that carry the corresponding inserted ‘worst-case’ target in the FRMD7 locus. d Genome editing efficiency of gRNAs adjacent to a disease-relevant ClinVar site and predicted to be self-complementary and to have very low cleavage efficiency (Doench 2016 and Moreno-Mateos percentile score both ≤5). Compared are t0 (normal), t0 (IDT) with proprietary chemical modifications, and GOLD tracrRNA. Cas9 RNPs were electroporated into 409-B2 hiPSCs. Independent biological replicates ( n = 3) are depicted by black dots and the error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: We used a human induced pluripotent stem cell (hiPSC) line (409-B2) carrying a doxycycline-inducible Cas9 (iCRISPR-Cas9) gene to test the efficiency of normal tracrRNA (t0), the locked tracrRNA (t-lock), and the normal tracrRNA with proprietary chemical modifications (t0-IDT) from a commercial provider in combination with ten different crRNAs, most of which have predicted strong non-canonical interactions to different regions in the gRNA (Fig. ).

Techniques: Sequencing, Introduce, Blocking Assay, Derivative Assay, Clone Assay, Expressing