Cheng Lab CRISPR Casilio Plasmids
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.
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.
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 platform website: Find information and resources on using Casilio, as well as the latest sgRNA-PBS sequences and modules
- JAX press release on the Casilio platform
For Cas9, use a nuclease-deficient S. pyogenes Cas9 with D10A and H840A mutations (dCas9). Addgene has many dCas9 plasmids available.
- pAC1445 – pmax-dCas9– A transient expression vector for dCas9.
A single-guide RNA (sgRNA) appended with multiple copies of Pumilio binding sites (PBS).
|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|
A fusion protein consisting of effector domain(s) fused with a PUF domain programmed to recognize a specific 8-mer PBS.
|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|