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)).
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Heterologous Expression of Cas13 Systems
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Variable analysis
independent variables
- EsCas13d (catalog 108303)
- CasRX (catalog 109049)
- PspCas13b (catalog 103862)
- Expression levels of Cas13 proteins monitored by western blotting
- Glutamate dehydrogenase promoter used to drive Cas13 expression
- PPAC-3HA expression cassettes used to express Cas13 proteins
- SwaI used to linearize Cas13-3HA expression vectors before electroporation into G. lamblia
- DCas9g1pac used as a backbone for generating the CasRX expression system
- DCas9 replaced with CasRX in the CRISPRi expression vector
- GRNA scaffold sequence replaced with the CasRX-specific direct repeat
- Not mentioned
- Not mentioned
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