CRISPi Kit
(Kit # 1000000136 )

Depositing Lab:   Brigitte Gasser

The CRISPi Kit contains basic elements for performing CRISPR/Cas9 mediated gene and genome editing in the yeast P. pastoris.

This kit will be sent as nine individual bacterial stabs.

Original Publication

CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris. Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer,R. Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9. PubMed Article

Additional References

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, Sauer M, Mattanovich D, Gasser B, Marx H. BMC Syst Biol. 2017 11,123. doi: 10.1186/s12918-017-0492-3. PubMed Article

Description

The CRISPi Kit contains basic elements for performing CRISPR/Cas9 mediated gene and genome editing in the yeast P. pastoris. The kit contains seven different backbone 3 (BB3) vectors with functional Cas9 and sgRNA expression cassettes and three different selection markers.

Each CRISPi BB3 carries:

• S. cerevisiae CEN6/ARS4 sequence for episomal plasmid maintenance, enabling the recovery of plasmid free cells after successful genome engineering
• Human codon-optimized hCas9 gene fused to a nuclear localization signal (NLS) under control of a constitutive promoter (original Cas9 sequence - Addgene #43802)
• An insertion site for the sgRNA with flanking ribozyme sequences under control of the P. pastoris PGAP promoter
• A marker cassette for yeast/E. coli selection natMX (nourseothricin (NTC) selection), hphMX (hygromycin B) or kanMX (geneticin (G418))

All plasmids were assembled using the GoldenPiCS Kit (Addgene Kit #1000000133).

Single guide (sg) RNA construction and assembly into CRISPi plasmids. (A) sgRNA constructs are assembled by overlap extension (OE) PCR of 6 overlapping primers. The variable sequence is covered by two primers (1_sgRNA_fw and 2_sgRNA_fw) and the structural part containing sgRNA and flanking ribozymes is made up by four primers (D_sgRNA_struc_fw and A, B and C_sgRNA_struc_rev). (B) The final HH-sgRNA-HDV construct is flanked by BpiI restriction sites with open fusion site Fs2 (5´) and Fs3 (3´). (C) CRISPi plasmids harbor all elements required for hCas9 and sgRNA expression in P. pastoris, HH–sgRNA–HDV and an empty HH–sgRNA–HDV expression cassette with internal BpiI sites between the sgRNA promoter and terminator (Fs 2,3 linker), allowing direct assembly of the final plasmids (D). (E) Final plasmids feature a CEN6/ARS4 sequence for episomal expression, a resistance marker for positive selection in E. coli and P. pastoris, an expression unit for the hCas9 (3 different promoters PPFK300, PLAT1 and PScTEF1 available) and the expression unit for the HH–sgRNA–HDV unit under control of the PGAP promoter and the TRPS25A terminator.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The CRISPi Kit was a gift from Brigitte Gasser (Addgene Kit #1000000136).”

For your Reference section:

CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris. Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer,R. Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9.

Kit Contents

Please refer to the individual plasmid page for more details on each stab in this kit:

ID	Name
104906	BB3cN_pGAP_23*_pLAT1_Cas9
104907	BB3cH_pGAP_23*_pLAT1_Cas9
104908	BB3cK_pGAP_23*_pLAT1_Cas9
104909	BB3cK_pGAP_23*_pTEF_Cas9
104910	BB3cK_pGAP_23*_pPFK300_Cas9
104911	BB3cN_pGAP_23*_pPFK300_Cas9
104912	BB3cH_pGAP_23*_pPFK300_Cas9
108686	BB3nK_ext_AC
108687	BB3nK_ext_AD