Pspcas13b

Manufactured by Addgene

PspCas13b is a CRISPR-Cas13 system derived from the Prevotella and Bacteroides species. It is a RNA-guided RNA-targeting effector that can be used for RNA interference and knock-down applications.

Automatically generated - may contain errors

Lab products found in correlation

Escas13d, by Addgene (3 mentions) Lwacas13a, by Addgene (2 mentions) Casrx, by Addgene (2 mentions) Pcdna3, by GenScript (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions)

Spelling variants of this product

PC0043-PspCas13b crRNA backbone (4 citations) PU6-PspCas13b-gRNA (4 citations) PC0046-EF1a-PspCas13b-NES-HIV (3 citations)

5 protocols using pspcas13b

Diverse Cas13 Variants for CRISPR Applications

Cited 2 times

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Cas13bt, EsCas13d, RspCas13d and PspCas13b were obtained from Addgene (#176316, #108303, #108305, and #103862, respectively)^{7 (link),16 (link),23 (link)}. EsCas13d, RspCas13d, and PspCas13b sequences were amplified by PCR and cloned into a pcDNA3.1 backbone. Cas13X (#171379) and REPAIRx were gifts from Professor Hui Yang and Xingxu Huang, respectively^{15 (link),38 (link)}. Mini-RfxCas13d, mini-EsCas13d, mini-RspCas13d, mini-PspCas13b, mini-PbuCas13b, PbuCas13b and mini-Vx were synthesized and cloned into pcDNA3.1(+) (GenScript Biotech, China). The Δ1, Δ2, and Δ3 RfxCas13d strains were constructed by homologous recombination. Δ4, Δ5, Δ6, Δ7, and Δ8 were synthesized and cloned into pcDNA3.1(+) by GenScript Biotech (China). The sequences of the proteins are listed in Table S1. All of the primers used for plasmid construction are listed in Table S2.

Zhao F., Zhang T., Sun X., Zhang X., Chen L., Wang H., Li J., Fan P., Lai L., Sui T, & Li Z. (2023). A strategy for Cas13 miniaturization based on the structure and AlphaFold. Nature Communications, 14, 5545.

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Plasmid Construction for CRISPR Tools

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Plasmids used in this study were prepared by standard molecular biology techniques and coding sequences entirely verified. Plasmids encoding LwaCas13a (#91902), PspCas13b (#103862), RfxCas13d (#109049), LwaCas13a crRNA backbone (#91906), RfxCas13d crRNA backbone (#109053), and Cas9&gRNA (#52961) were purchased from Addgene. The backbone expressing PspCas13b crRNA was engineered from #103862. For the construction of the plasmids encoding SIK3-S, SIK3-S-K37M, tdTomato, ZAKα, ZAKβ and NeuN, and AAV-PHP.eB backbone, the target fragments were amplified by PCR and ligated into the linearized corresponding vectors obtained by restriction enzyme (NEB) digestion via Gibson assembly (CL116-02, Biomed). For the construction of the plasmids encoding crRNAs, the primers for crRNA/gRNA were annealed and ligated into the linearized corresponding vectors cut by Bbs1 /BsmB1 (R0539S/R0739S, NEB) via T4 DNA ligase (M0202S, NEB). All plasmids were confirmed by sequencing. The sources, sequences, and application locations of all plasmids used were provided in Additional file 5: Table S4. In vitro-synthesized crRNAs were purchased from GenScript. All primers were order from Synbio Technologies or RuiBiotech. All sequencings were done in RuiBiotech.

Li Y., Xu J., Guo X., Li Z., Cao L., Liu S., Guo Y., Wang G., Luo Y., Zhang Z., Wei X., Zhao Y., Liu T., Wang X., Xia H., Kuang M., Guo Q., Li J., Chen L., Wang Y., Li Q., Wang F., Liu Q, & You F. (2023). The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model. Genome Biology, 24, 20.

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Heterologous Expression of Cas13 Systems

Cited 1 time

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EsCas13d (catalog 108303), CasRX (catalog 109049), and PspCas13b (catalog 103862) were obtained from Addgene. Cas13 fragments were PCR amplified, digested with restriction enzymes, and inserted into pPAC-3HA expression cassettes under the glutamate dehydrogenase promoter (54 (link)). The Cas13-3HA expression vectors were linearized with SwaI and electroporated into G. lamblia so that expression levels could be monitored by western blotting. To generate the CasRX expression system, the CRISPRi expression vector (dCas9g1pac) was used as a backbone, dCas9 was replaced with CasRX, and the gRNA scaffold sequence was replaced with the CasRX-specific direct repeat (36 (link)).

Shih H.W., Alas G.C, & Paredez A.R. (2022). A cell-cycle–dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia. Proceedings of the National Academy of Sciences of the United States of America, 119(22), e2204402119.

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CRISPR RNA Interference Systems

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Human cells LwaCas13a, PspCas13b, and CasRx expression plasmids were acquired from Addgene. Human cell U6 promoter expression plasmids for crRNA targeted Gaussia mRNA were generated by annealing and ligating the oligonucleotide duplexes into BbsI-digested LwaCas13a guide expression backbone, PspCas13b crRNA backbone, and CasRx pre-gRNA backbone vector, respectively. And the crRNA targeted endogenous genes KRAS, CXCR4, and PPIB were cloned into LwaCas13a guide expression backbone. The Gaussia and Firefly luciferase reporter plasmids were purchased from ThermoFisher Scientific. Human codon optimized AcrVIA sequences with a C-terminal nuclear localization signal (SV40) were cloned into pcDNA3.1+ via Gibson cloning.

Lin P., Qin S., Pu Q., Wang Z., Wu Q., Gao P., Schettler J., Guo K., Li R., Li G., Huang C., Wei Y., Gao G.F., Jiang J, & Wu M. (2020). CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells. Molecular cell, 78(5), 850-861.e5.

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Establishment of Cas13 Expression Systems in Giardia

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EsCas13d (Catalog #108303), CasRX (Cat #109049), and PspCas13b (Cat#103862) were obtained from Addgene. Cas 13 fragments were PCR amplified, digested with restriction enzymes and inserted into pPAC-3HA expression cassettes under GDH promoter 45 (link) . The Cas13-3HA expression vectors were linearized with SwaI and electroporated into Giardia lamblia. To generate CasRX expression system, CRISPRi expression vector (dCas9g1pac) was used as a backbone.

Shih H., Alas G.C.M., & Paredez A.R. (2021). A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia.

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