Pc0043 pspcas13b crrna backbone

Manufactured by Addgene

The PC0043-PspCas13b crRNA backbone is a genetic construct used in CRISPR-based gene editing systems. It provides a standardized template for the production of crRNA (CRISPR RNA) molecules, which are essential components of the Cas13b endonuclease system. The core function of this product is to facilitate the generation of customized crRNA sequences for targeted RNA manipulation and knockdown applications.

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Lab products found in correlation

T4 dna ligase, by Elpis Biotech (1 mentions) Ac5 stable2 neo, by Addgene (1 mentions) Pcfd5, by Addgene (1 mentions) Pc0046 ef1a pspcas13b nes hiv, by Addgene (1 mentions) Pcfd3, by Addgene (1 mentions) Pdsred attp, by Addgene (1 mentions) Pc0040 lwacas13a crrna backbone, by Addgene (1 mentions) Opti mem 1 reduced serum media, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

4 protocols using pc0043 pspcas13b crrna backbone

Site-Specific Methylation of lncRNAs

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Site-specific methylation of lncRNAs in cells was performed as described before [26 (link)]. Briefly the DNA fragment encoding the guide RNA targeting A1225 of PSMA3-AS1 (5′-TACAGGTTATCTCAGAATATCTTTTTTGGC-3′) or A2041 of MIR22HG (5′-ATATTGGGTCTTATTTTTTTCTGTTATGGT-3′) was inserted into pC0043-PspCas13b crRNA backbone (Addgene). Subsequently the recombinant pC0043-PspCas13b crRNA backbone was cotransfected with the plasmid expressing METTL3 fused with catalytically inactivated Cas13 (dCas13b; Addgene) into cells (10⁶/well) using Lipofectamine 2000 (Invitrogen). The cells were cultured in Opti-MEM I Reduced Serum Media (Gibco). The cells were harvested 36 h post-transfection.

Ci Y., Zhang Y, & Zhang X. (2024). Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA–miRNA/protein axis. Cellular & Molecular Biology Letters, 29, 51.

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CRISPR-Cas13 and Base Editor Plasmids for Gene Repair

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For the REPAIRv2 system we used the following plasmids:

pC0055-CMV-dPspCas13b-GS-ADAR2DD(E488Q/T375G)-delta-984-1090, from Addgene (RRID: Addgene_103871);

pC0043-PspCas13b crRNA backbone, from Addgene (RRID: Addgene_103854);

CFTR-gRNA W1282X 50–32 and CFTR-gRNA W1282X 50-34, designed by us [22 (link)].

For the mxABE system we used the following plasmids:

U6-BbsI-DR_CMV-minidCas13X.1-REPAIRv2-BGHpA_CMV-EGFP-BGHpA, from Addgene (RRID: Addgene_171383);

mxABE-NT, coding for a control gRNA (non-targeting) (Figure S2);

mxABE-CFW1282X 50–25, coding for a gRNA specific for the W1282X mutant region, 50 nts long, with a mismatch in position 25 with respect to 5′end (Figure S2);

mxABE-CFG542X 50–25, coding for a gRNA specific for the G542X mutant region, 50 nts long, with a mismatch in position 25 with respect to 5′end (Figure S2).

Chiavetta R.F., Titoli S., Barra V., Cancemi P., Melfi R, & Di Leonardo A. (2023). Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells. International Journal of Molecular Sciences, 24(13), 10940.

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Generating crRNA Targeting Diverse Genes

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The crRNAs targeting the Fluc reporter, the GFP indicator, NRAS, NFKB1, PPARG, KRAS, PPIB, STAT3, and the A-to-I or C-to-U RNA editing sites were generated by annealed oligo cloning using the BbsІ site of the pC0043-PspCas13b crRNA backbone (Addgene plasmid 103854). All crRNA plasmids were cloned using T4 DNA ligase (Elpis Biotech).

Yu J., Shin J., Yu J., Kim J., Yu D, & Heo W.D. (2024). Programmable RNA base editing with photoactivatable CRISPR-Cas13. Nature Communications, 15, 673.

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Comprehensive Plasmid Database for Genetic Manipulation

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For a list of plasmids, we generated for this study, see Additional file 3: Table S2. The original plasmids we used for this project were obtained from Addgene: pCFD3 (#49410), pCFD5 (#73914), pACG:eCFP (#32597), pDsRed-attP (#51019), Ac5-Stable2-Neo (#32426), pC0056-LwaCas13a-msfGFP-NES (#105815), pC0040-LwaCas13a crRNA backbone (#103851), pC0046-EF1a-PspCas13b-NES-HIV (#103862), pC0043-PspCas13b crRNA backbone (#103854), pC0054-CMV-dPspCas13b-longlinker-ADAR2DD (E488Q/T375G) (103870), pXR001: EF1-CasRX-2A-eGFP (#109049), pXR004: CasRX pre-gRNA cloning backbone (#109054), pBID-UASc (#35200), [10 (link), 12 (link), 23 (link), 35 (link), 46 (link), 50 (link), 107 (link)–110 (link)]. We also obtained plasmids from the Drosophila Genetic Resource Center (DGRC): pAFW (#1111), pAHW (#1095), act-PhiC31-integrase (#1368). We also used plasmids we previously generated, enDmC, to generate some constructs for this study [8 (link)]. pMT-Gal4-puro plasmid was a kind gift from Christoph Metzendorf (University of Uppsala). All fragments used for the cloning step were amplified via PCR using Q5 high-fidelity DNA polymerase (NEB #M0491S) (Additional file 4: Table S3) and fused together via Gibson assembly reaction [111 (link)].

Huynh N., Depner N., Larson R, & King-Jones K. (2020). A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biology, 21, 279.

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