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, respectively15 (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 .
Escas13d
Manufactured by Addgene
EsCas13d is a CRISPR-Cas13d system derived from Eubacterium siraeum. Cas13d is a class 2 CRISPR effector that can be programmed to target and cleave RNA sequences. The EsCas13d system allows for RNA targeting and manipulation in research applications.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using escas13d
1
Diverse Cas13 Variants for CRISPR Applications
2
Heterologous Expression of Cas13 Systems
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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)).
3
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.
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