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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 the Cbh promoter with EF1alpha
  • addition of an 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

Plasmids 159654 and 159655 contain a U6 promoter, gRNA scaffold, EF1alpha promoter, Cas9-T2A-H2B-GFP (or mCherry). Plasmids 171098 and 175570 are donor plasmids to add an additional U6-gRNA scaffold to the pX458 plasmid via digestion with XbaI and KpnI. Plasmids repression plasmids 175573 and 175574 contain a U6 promoter, gRNA scaffold, EF1alpha promoter, Cas9-KRAB-MeCP2-T2A-H2B-GFP (or mCherry). Activation plasmids 175571 and 175572 contain a U6 promoter, gRNA scaffold, EF1alpha promoter, Cas9-VPR-T2A-H2B-GFP (or mCherry).
Figure 1: 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:
Top strand oligos need a 5’ CACCG overhang. Bottom strand oligos should have a 5’ AAAC and 3’C.
  • 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:
Sequences show BbsI recognition and cut sites, to show how the overhangs are created. BbsI recognition site is GTCTTC and cuts six nucleotides upstream with a four nucleotide overhang. Two BbsI sites are next to each other to create the two overhangs for the oligonucleotides.
  • 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

Plasmid and Sequence Derivative Sources

Plasmids from Collection

ID Plasmid Description PI

Publications

  • Kaufman, M. L., Goodson, N. B., Park, K. U., Schwanke, M., Office, E., Schneider, S. R., Abraham, J., Hensley, A., Jones, K. L., & Brzezinski, J. A. (2021). Initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity. Development, 148(12). doi: 10.1242/dev.199399. PMID: 34143204
  • Goodson, N. B., Kaufman, M. A., Park, K. U., & Brzezinski, J. A. (2020). Simultaneous deletion of Prdm1 and Vsx2 enhancers in the retina alters photoreceptor and bipolar cell fate specification, yet differs from deleting both genes. Development. doi: 10.1242/dev.190272. PMID: 32541005