This website uses cookies to ensure you get the best experience. By continuing the use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at help@addgene.org. Learn more

Casilio logo Cheng Lab CRISPR Casilio Plasmids


Background

The Albert Cheng and Haoyi Wang labs at The Jackson Laboratory (JAX) have developed a CRISPR/Cas platform called Casilio , which is a hybrid system combining d Cas 9 and the RNA-binding domain from Pum ilio . The system uses a modified sgRNA, referred to as sgRNA-PBS, that has different numbers of 8-mer P UF b inding s ites (PBS). These PUF-binding sites recruit PUF (Pumilio/FBF) domains that are fused to an effector, like those for epigenetic regulation, chromosomal labeling, and gene regulation. This is an alternate strategy to techniques that fuse effector domains to dCas9.

Casilio schematic

PUF can be programmed to recognize virtually any 8-mer RNA sequences. This feature enables Casilio to multiplex and mulitmerize effectors at distinct loci. It can also potentially recruit different proteins on a single sgRNA allowing the study of protein complexes. Due to the 8-mer RNA sequence space provided by the PUF code, it is possible to construct approximately 60,000 orthogonal modules that can function independently in cells. In the future, modules tethering different fusion proteins can be assigned a unique PUF/PBS pairing. The Cheng lab will maintain a Casilio module database with links to resources and information. Researchers can obtain the desired modules to perform their functional genomic experiments by constructing the matching sgRNA-PBS. Since Casilio modules are orthogonal, researchers can use any combinations of modules as is, allowing plug-and-play application of Casilio in functional genomics studies.

casilio system diagram showing progression from database to module design
Overview of Casilio workflow. The Cheng Lab is maintaining a Casilio website containing a module database where researchers can search for available modules, as well as find information on the Casilio platform.

Protocols & References

Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cheng AW, Jillette N, Lee P, Plaskon D, Fujiwara Y, Wang W, Taghbalout A, Wang H. Cell Res. 2016 Feb;26(2):254-7. Open Access Link. PubMed PMID: 26768771 .

Casilio Plasmids

Cas9 plasmids

For Cas9, use a nuclease-deficient S. pyogenes Cas9 with D10A and H840A mutations (dCas9). Addgene has many dCas9 plasmids available.

sgRNA-PBS plasmids

A single-guide RNA (sgRNA) appended with multiple copies of Pumilio binding sites (PBS).

ID Name Description
71888 pAC1371-pX-sgRNA-5xPBSa Cloning vector for expression of sgRNA-5xPBSa
71889 pAC1372-pX-sgRNA-15xPBSa Cloning vector for expression of sgRNA-15xPBSa
71890 pAC1373-pX-sgRNA-25xPBSa Cloning vector for expression of sgRNA-25xPBSa
71891 pAC1374-pX-sgRNA-5xPBSb Cloning vector for transient expression of sgRNA-5xPBSb
71892 pAC1375-pX-sgRNA-15xPBSb Cloning vector for expression of sgRNA-15xPBSb
71893 pAC1376-pX-sgRNA-25xPBSb Cloning vector for expression of sgRNA-25xPBSb
71894 pAC1379-pX-sgRNA-5xPBSw Cloning vector for expression of sgRNA-5xPBSw
71895 pAC1380-pX-sgRNA-5xPBSc Cloning vector for expression of sgRNA-5xPBSc
71898 pAC1394-pX-sgRNA-0xPBS Cloning vector for expression of sgRNA without PBS. It contains extra sequences for BsaI digestion for insertion of PBS
71899 pAC1399-pX-sgRNA-20xPBSc Cloning vector for expression of sgRNA-20xPBSc
71917 pAC1420-pX-sgRNA-1xPBSa Cloning vector for expression of sgRNA-1xPBSa
71918 pAC1421-pX-sgRNA-2xPBSa Cloning vector for expression of sgRNA-2xPBSa
71919 pAC1422-pX-sgRNA-1xPBSb Cloning vector for expression of sgRNA-1xPBSb
71920 pAC1423-pX-sgRNA-2xPBSb Cloning vector for expression of sgRNA-2xPBSb
71921 pAC1424-pX-sgRNA-10xPBSb Cloning vector for expression of sgRNA-10xPBSb
71925 pAC1425-pX-sgRNA-20xPBSb Cloning vector for expression of sgRNA-20xPBSb
71926 pAC1426-pX-sgRNA-47xPBSb Cloning vector for expression of sgRNA-47xPBSb
71927 pAC1427-pX-sgRNA-10xPBSw Cloning vector for expression of sgRNA-10xPBSw
71928 pAC1428-pX-sgRNA-15xPBSw Cloning vector for expression of sgRNA-15xPBSw
71929 pAC1429-pX-sgRNA-10xPBSc Cloning vector for expression of sgRNA-10xPBSc
71930 pAC1430-pX-sgRNA-15xPBSc Cloning vector for expression of sgRNA-15xPBSc

Casilio modules

A fusion protein consisting of effector domain(s) fused with a PUF domain programmed to recognize a specific 8-mer PBS.

ID Name Description
71881 pAC1355-pmax-NLSPUFa_VP64 NLSPUFa_VP64 in transient expression vector pmax
71882 pAC1356-pmax-NLSPUFb_VP64 NLSPUFb_VP64 in expression vector pmax
71883 pAC1357-pmax-NLSPUFw_VP64 NLSPUFw_VP64 in expression vector pmax
71884 pAC1358-pmax-NLSPUFc_VP64 NLSPUFc_VP64 in expression vector pmax
71897 pAC1393-pmax-NLSPUFa_p65HSF1 NLSPUFa_p65HSF1 in pmax expression vector
71900 pAC1402-pCR8-Clover_NLSPUFa Clover_NLSPUFa in pCR8 gateway donor vector
71901 pAC1403-pCR8-Clover_NLSPUFc Clover_NLSPUFc in pCR8 gateway donor vector
71902 pAC1404-pCR8-mRuby2_NLSPUFa mRuby2_NLSPUFa in pCR8 gateway donor vector
71903 pAC1405-pCR8-4xNLS_PUFa_2xNLS NLSPUFa pCR8 gateway donor vector for insertion of N-terminal domain (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest
71904 pAC1406-pCR8-4xNLS_PUFb_2xNLS NLSPUFb pCR8 gateway donor vector for insertion of N-terminal domain (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest
71905 pAC1407-pCR8-4xNLS_PUFw_2xNLS NLSPUFw pCR8 gateway donor vector for insertion of N-terminal domain (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest
71906 pAC1408-pCR8-4xNLS_PUFc_2xNLS NLSPUFc pCR8 gateway donor vector for insertion of N-terminal domain (SgrAI or AgeI with ClaI) and C-terminal domain (FseI PacI). Grow in dcm- cells (e.g., NEB C3040) to prepare vector for ClaI digest
71908 pAC1411-pmax-NLSPUFc_p65HSF1 NLSPUFc_p65HSF1 in pmax expression vector
71912 pAC1415-pCR8-4xNLS_PUFa_2xNLS_mCBPHAT 4xNLS_PUFa_2xNLS_mCBPHAT in pCR8 Gateway donor vector
71913 pAC1416-pCR8-mCBPHAT_4xNLS_PUFa_2xNLS mCBPHAT_4xNLS_PUFa_2xNLS in pCR8 Gateway donor vector
71914 pAC1417-pmax-4xNLS_PUFa_2xNLS_mCBPHAT 4xNLS_PUFa_2xNLS_mCBPHAT in pmax expression vector
71915 pAC1418-pmax-mCBPHAT_4xNLS_PUFa_2xNLS mCBPHAT_4xNLS_PUFa_2xNLS in pmax expression vector