Brzezinski Lab CRISPR Collection

The Brzezinski lab investigates gene regulation in the context of the developing mouse retina. To aide in their studies of the regulatory networks involved in retinal development the lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate gene targets. To accomplish this they have taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several modifications to the the px458 plasmid to expand its usefulness for the lab's gene regulation and developmental studies.

Key plasmid features and pX458 modifications:

• replacement of Cbh promoter with EF1alpha
• addition of mCherry reporter variant
• nuclear reporters via an H2B fusion
• shuttle plasmid for shuttling U6-guide cassettes to make dual guide expressing plasmids
• dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression
• dCas9-VPR fusion for gene activation

CRISPR/Cas9 Plasmid Variants

Schematic of the CRISPR Collection. Plasmids 159654 and 159655 are single guide expressing double stranded break constructs. Plasmids 171098 and 175570 allow conversion to a dual guide construct. Plasmids 175573 and 175574 express dCas9-KRAB-MeCP2 for repression. Plasmids 175571 and 175572 express dCas9-VPR for activation.

Usage

• Guide sequences can be designed using Benchling or using similar design tools and ordered as standard desalted oligonucleotides with the appropriate overhangs:
  

  

• The plasmids listed here are provided as empty backbones that utilize Golden Gate cloning to insert the desired guide RNA sequence by digestion of the backbone with BbsI followed by a quick ligation of annealed and phosphorylated oligonucleotides containing the guide target sequence:
  

  

• Up to two guides can be expressed from the same plasmid by utilizing one of the dual guide shuttle plasmids to insert a second guide within a U6-guide cassette. This process is compatible with all pX458 family plasmids.

Additional Cloning Information

Cloning Protocols

• Single guide cloning: Zhang Lab General Cloning Protocol 237.2 KB
• Dual guide cloning: Brzezinski Lab Dual Guide Protocol 967.2 KB

Plasmid and Sequence Derivative Sources

• Original pX458 backbone (Addgene #48138)
• dCas9 + KRAB-MeCP2 (Addgene #110821)
• VPR (Addgene #63798)

Plasmids from Collection

Publications

• Kaufman ML, Goodson NB, Park KU, Schwanke M, Office E, Schneider SR, Abraham J, Hensley A, Jones KL, Brzezinski JA Development. 2021 Jun 15;148(12). pii: 269198. doi: 10.1242/dev.199399. Epub 2021 Jun 18. PMID 34143204
• Goodson NB, Kaufman MA, Park KU, Brzezinski JA 4th Development. 2020 Jul 3;147(13). pii: dev.190272. doi: 10.1242/dev.190272. PMID 32541005