rudolf jaenisch  (Addgene inc)

Journal: bioRxiv

Article Title: Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

doi: 10.1101/2023.05.08.539738

Figure Lengend Snippet: First generation iSBH-sgRNAs detect short RNA triggers in HEK293T cells. A . Native sgRNA sequences are composed of spacer and scaffold sequences . iSBH-sgRNAs fold into complex secondary structures that interfere with the Cas9 ability to recognise target DNA sequences (OFF-state, ). iSBH-sgRNAs were designed by extending the 5’ end of the spacer sequence with a 14nt loop and a spacer* sequence partially complementary with the spacer. Bulges were also introduced within the iSBH-sgRNA sequence in order to ensure that the interaction between the spacer* and RNA trigger is more energetically favourable. In the ON-state, iSBH-sgRNAs recognise complementary RNA triggers and become activated, enabling Cas9 to perform its function. Short RNA triggers are complementary with the iSBH-sgRNA loop and spacer* sequence. B . Inside cells, RNA triggers are expected to bind to complementary iSBH-sgRNAs, inducing iSBH-sgRNA activation. Activated iSBH-sgRNAs are recognised by CRISPRa effectors and drive ECFP production from a fluorescent reporter. In this particular example, activated iSBH-sgRNAs interact with dCas9-Vp64 and drive ECFP production from an 8xCTS-ECFP reporter . Following reporter induction, ECFP production could be monitored by Flow Cytometry. C . Starting from five different sgRNA spacer sequences, we designed 5 different iSBH-sgRNA sequences. For each iSBH-sgRNA, corresponding RNA triggers and 8xCTS-ECFP reporters were also designed. Ability of first-generation iSBH-sgRNA designs to drive expression of the ECFP reporter was assessed in the absence or presence of complementary RNA triggers. Experiments were carried out using dCas9-Vp64 and 8xCTS-ECFP reporters. D . An orthogonality test was performed, in which the 5 iSBH-sgRNA designs were tested against all 5 RNA triggers. Activation is only detected in the presence of matching iSBH-sgRNA and RNA trigger pairs. Figure shows mean +/-standard deviation values measured for 3 biological replicates. P-values were determined through unpaired t-tests. Figure 1—figure supplement 1 . First generation iSBH-sgRNAs detect short RNA triggers in HEK293T cells.

Article Snippet: 8xCTS sequences were cloned in the P2-ECFP-pA (Addgene #26280) plasmid generated by Nissim et al ( ).

Techniques: Sequencing, Activation Assay, Flow Cytometry, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

doi: 10.1101/2023.05.08.539738

Figure Lengend Snippet: Modular iSBH-sgRNA designs enable spatial separation of spacer and trigger-sensing sequences. A . In second-generation iSBH-sgRNAs, RNA triggers are complementary with the iSBH-sgRNA backfolds, thus sgRNA spacers influence RNA trigger sequences. In modular iSBH-sgRNAs, design constrains were eliminated as triggers are only complementary with the iSBH-sgRNA loop and first 15nt of the backfold. To increase affinity between iSBH-sgRNAs and RNA triggers, we increased loop sizes. Separation between trigger-sensing and spacer sequences was also achieved by reducing the complementary between the spacer sequence and CTS from 20 to 17nt. B . MODesign enables users to design modular iSBH-sgRNAs starting from input RNA triggers, sgRNA spacers and loop sizes. MODesign calculates the size of trigger-sensing sequences and creates a list of trigger sub-sequences having that size. Script determines the reverse complement of these sequences that could act as trigger-sensing sequences. iSBH-sgRNAs are assembled through adding spacer*, trigger-sensing sequences, extension, spacer and scaffold sequences. Extension sequences are engineered to be partially complementary with trigger-sensing sequences. Before producing a list of output sequences, iSBH-sgRNA folding is checked using NuPACK . Simulations could result in multiple modular iSBH-sgRNA designs. Designs chosen for experimental validation were selected based on the probability of folding into the iSBH-sgRNA structure and lack of trigger secondary structures in the iSBH-sgRNA complementary region. Priority was also given to iSBH-sgRNAs that, by chance, displayed extra complementarity between RNA triggers and the last 15nt of the backfold or more than 17nt complementarity with the CTS. C . MODesign simulations were carried out for designing iSBH-sgRNAs capable of sensing trigger RNA D (146nt eRNA sequence). In each simulation, a different sgRNA sequence was used and a desired loop size of 14nt was kept constant between simulations. Selected designs were transfected to HEK293T cells together with the RNA trigger D sequence (expressed from a U6 promoter). Tests were carried out using dCas9-Vp64 and 8xCTS-ECFP reporters. D . MODesign simulations were run for designing iSBH-sgRNAs capable of sensing trigger RNA A (146nt repetitive RNA sequence), trigger RNA B (267nt repetitive RNA sequence), trigger RNA C (268nt repetitive RNA sequence) and trigger RNA D (146nt eRNA sequence). Tests were performed using different CRISPRa effectors. E . 4 modular iSBH-sgRNAs (A,B,C and D) were co-transfected to HEK293T cells and all iSBH-sgRNA: RNA trigger combinations were tested. Figure shows mean +/-standard deviation values measured for 3 biological replicates. P-values were determined through unpaired t-tests. Figure 3—figure supplement 1 . Modular iSBH-sgRNA designs enable spatial separation of spacer and trigger-sensing sequences.

Article Snippet: 8xCTS sequences were cloned in the P2-ECFP-pA (Addgene #26280) plasmid generated by Nissim et al ( ).

Techniques: Sequencing, Biomarker Discovery, Transfection, Standard Deviation

Journal: bioRxiv

Article Title: Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

doi: 10.1101/2023.05.08.539738

Figure Lengend Snippet: Testing the ability of second generation iSBH-sgRNA designs to detect short RNA triggers in vivo . A . Transgenic lines encoding dCas9-Vp64 and 8xCTS-ECFP reporters were created. Embryos resulting from in crossing first generation (F1) transgenics were injected with second generation chemically synthesised iSBH-sgRNAs and RNA triggers. B . Second-generation iSBH-sgRNAs were injected into transgenic zebrafish embryos with or without corresponding short RNA triggers. In the absence of RNA triggers (iSBH-sgRNA OFF), embryos are expected to display no ECFP signals, while trigger presence (iSBH-sgRNA ON) should promote ECFP expression. C . Figure presents our second strategy for chemically modifying iSBH-sgRNAs. This strategy involved protecting the iSBH-sgRNA 5’ end as well as the 5’end of the sgRNA spacer. These modifications were used together with sgRNA scaffold modifications used in the IDT sgRNA XT designs . D . In order to quantify the impact of RNA triggers on iSBH-sgRNA activation, we grouped fish according to the intensity of ECFP signals. At 3 days post-fertilisation, embryos displaying no, low or high ECFP expression were counted. E . Embryos injected with iSBH-sgRNAs and non-complementary (iSBH-sgRNA OFF) of complementary RNA triggers (iSBH-sgRNA ON) were scored according to their ECFP intensity. Row number counts determined for 3 experimental replicates are displayed as part of Chi 2 contingency tables. P values displayed were determined using Chi 2 test. F Figure shows percentage of embryos recovered in each category for the 3 experimental replicates. Percentage of embryos with no ECFP expression varied between the 3 experimental replicates. This was due to the fact that both 8xCTS-ECFP and dCas9-VP64 transgenes are necessary for successfully expressing ECFP. These alleles segregate in a Mendelian fashion and our adult transgenic fish encode variable copy numbers of the transgene. For each individual replicate, we used embryos with identical genetic backgrounds for testing the iSBH-sgRNA (OFF) and iSBH-sgRNA (ON) conditions. Nevertheless, genetic backgrounds were different between the 3 experimental replicates. Figure 5—figure supplement 1 . Optimising sgRNA delivery to zebrafish embryos. Figure 5—figure supplement 2 . Testing different iSBH-sgRNA chemical modifications in vivo .

Article Snippet: 8xCTS sequences were cloned in the P2-ECFP-pA (Addgene #26280) plasmid generated by Nissim et al ( ).

Techniques: In Vivo, Transgenic Assay, Injection, Expressing, Activation Assay