CRISPR genome engineering
Genome engineering has advanced tremendously with the characterization of bacterial and archaeal CRISPR (clustered regularly interspaced short palindromic repeats) systems and their adapted usage in mammalian cells. The ability to precisely and permanently alter endogenous genomic sequence through targeted genome editing is a highly effective genetics tool.
In addition to the canonical Cas9 nuclease, other editing nucleases have been discovered including Cas12a (includes MAD7), Cas12b, Cas12c, Cas13, and many more.
Gene editing with MAD7 nuclease
MAD7 is a novel nuclease that was released by Inscripta in 2017. This nuclease only requires a crRNA for gene editing and allows for specific targeting of AT rich regions of the genome. MAD7 cleaves DNA with a staggered cut as compared to S. pyogenes which has blunt cutting.
Gene Editing with MAD7
The PAM sequence is YTTV. Y indicates a C or T base, and V indicates A, C or G. The PAM sequence is located upstream of the target sequence, and the repeat sequence appended to the 5’ of the target sequence is TTAATTTCTACTCTTGTAGAT.
The DNA cleavage sites for MAD7 relative to the target site are 19 bases after the YTTV PAM site on the sense strand and 23 bases after the ecomplementary PAM site of the anti-sense strand.
When designing experiments it is always important to include controls, the recommended crRNA control sequences for use in human cell lines are below:
| Human PPIB |
5’-UUAAUUUCUACUCUUGUAGAUACCUACGAAUUGGAGAUGAAG-3’ |
| Human DNMT3B |
5’-UUAAUUUCUACUCUUGUAGAUAAAUGAACCCUGCGCUGUCAU-3’ |
These can be easily ordered through the CRISPR design tool by selecting ‘Input my own guide RNA sequence’ from the Start tab.
The following synthetic crRNA products may be ordered through the CRISPR Design Tool. Contact Scientific Support if larger quantities are needed.
Synthetic crRNA is also available at a discounted price in 96-well plates, when ordering a minimum of 20 wells.
MAD7 can be used for editing in mammalian cells
MAD7, Cas9 or Cpf1-expressing plasmids (200 ng/well) were co-transfected with their respective synthetic guide RNAs (25 nM) into 293T cells using 0.7 µL/well of DharmaFECT Duo transfection reagent. PPIB (A) and DNMT3B (B) gene editing activities (estimated percentage of indels) were determined by a T7EI DNA mismatch detection assay.
Evaluation of MAD7 editing activity with ribonucleoprotein complex
MAD7, Cas9 proteins (25 nM) and their respective guide RNAs (50 nM) were co-transfected into HCT116 cells using 0.4 µL/well of DharmaFECT Duo transfection reagent. After 72 hours, gene editing activities (estimated percentage of indels) of PPIB (A) and STAG2 (B) were determined using T7EI DNA mismatch assay.
