What is a MADzyme? What does MADzyme mean?
Inscripta is developing new classes of CRISPR endonucleases, termed “MADzymes.” The MADzyme name is inspired by the biological diversity found on the island of Madagascar. MADzymes have improved features such as different PAM recognition sequences, different cut efficiencies, reduced sizes, and/or different enzyme kinetics. MAD7 is the first enzyme in this family that Inscripta has released.
What is MAD7?
MAD7 is a codon-optimized nuclease from theEubacterium rectale genome (refseq WP_055225123.1) This nuclease has 76% identity to the native Eubacterium rectale nucleotide sequence.
How similar is MAD7 to Cas9?
MAD7 is highly divergent from Cas9 in terms of structure, mechanism of action, and sequence (<25% aa. Identity.)
How similar/identical is MAD7 to Cpf1?
Although there are some structural similarities between MAD7 and the Cpf1 family, the Inscripta-engineered MAD7 is only 31% conserved with the canonical AsCpf1 from Acidominococcus sp. at the amino acid level.
How big is the enzyme?
MAD7 is a monomeric 147.9 kDa polypeptide consisting of 1263 amino acids (3782 nucleotides) in length.
What PAM site does MAD7 use?
This enzyme shows preference for TTTN and CTTN PAM sites.
What is the structure of the MAD7 gRNA?
Each guide RNA contains a 5’-GTCAAAAGACCTTTTTAATTTCTACTCTTGTAGAT sequence and should be designed to target 5’-YTTN-3’ PAMs using the first 21 nucleotides directly adjacent to the 3’ side of the PAM:
We recommend using a strong promoter to drive crRNA expression from a plasmid.
Where can I get data on performance?
Please download the poster and white paper at inscripta.com/madzymes/ for details
What expression system was used in your poster?
Codon-optimized MAD7 was cloned into a low-copy psc101 origin vector under control of the temperature-inducible pL promoter derived from the 𝝺 phage, and expressed in coli MG1655. In S. cerevisiae S288C, MAD7 was expressed from the RNR2 promoter and the gRNA was expressed from the SNR52 promoter.
What are the editing efficiencies in mammalian cells?
We are currently in the process of testing MAD7 in mammalian systems, and expect to present this data in the near future.
Have you measured off-target effects?
Targeting of unintended loci can result from many factors, including gRNA design, the hosts ability to repair cut dsDNA, and the host organism genome. We are in the process of evaluating MAD7 specificity in many organisms, including mammalian systems.
What is the off-target rate? How do you control for them?
We are currently in the process of testing MAD7 in mammalian systems, including off-targeting. There are a range of published methods for assessing and attempting to control for off-targeting.
Does MAD7 work for HDR-mediated precision editing?
Yes, we have performed precision editing using homology-directed repair in both coli and S. cerevisiae.
Do you have a dMAD7 available yet? Do you have a nicking MADzyme available?
We are currently working on MAD variants with altered functions, which we expect to make available in the future.
How does the performance of MAD7 compare against Cas9 and Cpf1?
While we have not performed head-to-head comparisons, we have observed editing efficiencies that are comparable to published results of other nucleases.
Does Inscripta have other collaborators that have used MAD7?
We have confirmed function of MAD enzymes through an academic collaboration. Inscripta is also interested with partnering with select academic and industrial research groups. If interested, please contact firstname.lastname@example.org
How free is MAD7, really?
Inscripta is providing the DNA sequence of MAD7 free for any R&D use. Please refer to the Terms for additional details.
Is it possible to obtain the physical MAD7 enzyme or a vector encoding MAD7?
Currently Inscripta is only providing the sequence of the enzyme. However, Inscripta is working with potential commercial partners to promote wide availability of the enzyme itself and vectors encoding the enzyme. We have had success synthesizing MADzymes from companies such as GeneArt and IDT.
How can I be sure my use in commercial research and development will not be subject to a future royalty?
Inscripta is committed to providing the MAD7 sequence for all R&D use without reach-through royalty rights, which means any use of the enzyme in the development of a commercial product will not result in a royalty to Inscripta for making, using or selling the developed commercial product. The only time a royalty will attach to the use of MAD7 is for a manufacturing process that uses MAD7 on an ongoing basis or a product that physically contains MAD7 or a related enzyme covered by Inscripta IP. Specific terms of the use of this enzyme can be found at inscripta.com/madzymes
Will Inscripta be releasing other MADzymes?
Inscripta is continuing its development of novel nucleic acid-guided endonucleases. Visit Inscripta.com for future releases.
Are custom MADzymes available for exclusive use?
In addition to MAD7, Inscripta is also engineering novel, bespoke enzymes for exclusive use by industry partners. Please contact us at email@example.com for more information.
How do I invest in your company?
Inscripta is a privately held company.
How do I get more information?
Please send inquiries and correspondence to firstname.lastname@example.org