Part:BBa_K2361001:Design
dCas9 VRER
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1100
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3379
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part originates from Streptococcus pyogenes Cas9 and it contains 7 mutations. Two amino acid mutations to catalitically inactivate it ( D10->A & H840->A) were already present in the part we started with. To remove the EcorI site an A->T substitution was made at position 1291, this mutation preserves the Ile codon. To change the PAM preference a set of four amino acid substitutions was performed (D1135V, G1218R, R1335E, T1337R). Besides the above mentioned mutations the spCas9 sequence was preserved.
Source
This part originates from the Addgene plasmid pJWV102-PL-dCas9. The VRER mutations, described for Cas9 by Heler et al., were added by replacing the last part of dCas9 with a piece of synthetic DNA.
References
Heler, R. et al. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response. Mol. Cell 65, 168–175 (2017)
High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW. Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.