The MAD7 nuclease: made for discovery.

WHATzyme nucleases?

Inspired by the biological diversity found on the island of Madagascar, the MADzyme nucleases have improved features over commonly used CRISPR-Cas9 nucleases, such as different PAM recognition sequences and cut efficiencies, fewer off-target effects, reduced sizes and differing enzyme kinetics. 

We developed and publicly released the MAD7 nuclease, the first of the MADzyme nucleases, to promote widespread adoption of CRISPR tools in academic and commercial settings. The MAD7 nuclease has already been shown to be effective in microbial, plant, fish and mammalian systems. Go ahead and try it, the MAD7 nuclease is royalty-free for both academic and commercial research and development use.

MAD7 nuclease structural model
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The MAD7 nuclease in mammalian cells

In addition to robust performance in prokaryotic and eukaryotic microbial systems, the MAD7 nuclease has demonstrated editing activity in mammalian cells. 

For example, initial experiments in HEK293T cells show that the MAD7 nuclease can be expressed as an active protein in mammalian cells, and when combined with synthetic guide RNAs, can edit different genes at multiple loci.

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Is the MAD7 nuclease really free?

For academic and commercial R&D, yes! Inscripta provides the DNA sequence of the E. coli codon-optimized MAD7 nuclease without reach-through royalty rights. Royalties are ONLY attached to the use of the MAD7 nuclease if a commercial manufacturing process uses the MAD7 nuclease on an ongoing basis or a product physically contains the MAD7 nuclease. A license is provided when the sequence is downloaded, and if requested, Inscripta can provide a hard copy license for free use of the MAD7 nuclease in R&D.

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The MAD7 nuclease FAQs

Inscripta is developing new classes of CRISPR endonucleases inspired by the biological diversity found on the island of Madagascar. The MADzyme nucleases have improved features such as different PAM recognition sequences and cut efficiencies, reduced sizes, and/​or different enzyme kinetics. The MAD7 nuclease is the first enzyme in this family that Inscripta has released.

The MAD7 nuclease is a codon-optimized nuclease from the Eubacterium rectale genome (refseq WP_055225123.1). This nuclease has 76% identity to the native Eubacterium rectale nucleotide sequence.

The MAD7 nuclease is highly divergent from Cas9 in terms of structure, mechanism of action, and sequence (<25% aa. identity).

Although there are some structural similarities between the MAD7 nuclease and the Cpf1 family, the Inscripta-engineered MAD7 nuclease is only 31% conserved with the canonical AsCpf1 from Acidominococcus sp. at the amino acid level.

While we have not performed head-to-head comparisons, we have observed editing efficiencies that are comparable to published results of other nucleases.

The MAD7 nuclease is a monomeric 147.9 kda polypeptide consisting of 1263 amino acids (3782 nucleotides) in length.

This enzyme shows preference for TTTN and CTTN PAM sites.

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:

5’ – GTCAAAAGACCTTTTTAATTTCTACTCTTGTAGAT-NNNNNNNNNNNNNNNNNNNNN.

We recommend using a strong promoter to drive crRNA expression from a plasmid.

Please download the poster and white paper at inscrip​ta​.com/​t​e​c​h​n​i​c​a​l​d​o​c​u​ments for details.

Codon-optimized MAD7 nuclease 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 E. coli MG1655. In S. cerevisiae S288C, the MAD7 nuclease was expressed from the RNR2 promoter and the gRNA was expressed from the SNR52 promoter.

We are currently in the process of testing the MAD7 nuclease in mammalian systems and expect to present this data in the near future.

Targeting of unintended loci can result from many factors, including gRNA design, the host’s ability to repair cut dsDNA, and the host organism genome. We are in the process of evaluating the MAD7 nuclease specificity in many organisms, including mammalian systems.

We are currently in the process of testing the MAD7 nuclease in mammalian systems, including measuring off-target effects. There are a range of published methods for assessing and attempting to control off-target effects.

Yes, we have performed precision editing using homology-directed repair in E.coli, S. cerevisiae, and mammalian cells.

We are currently working on MAD variants with altered functions, which we expect to make available in the future.

We have confirmed function of the MAD7 nuclease through academic and commercial collaborations. Inscripta is also interested in partnering with select academic and industrial research groups. If interested, please contact info@​inscripta.​com.

Currently Inscripta is only providing the sequence of the enzyme. However, we are working with potential commercial partners to promote wide availability of the enzyme itself and vectors encoding the enzyme. We have had success synthesizing the MADzyme nucleases from companies such as GeneArt and IDT. If you would like to have MAD7 nuclease produced by a third party please contact us at info@​inscripta.​com.

We’re committed to providing the MAD7 nuclease 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 the MAD7 nuclease is for a manufacturing process that uses the MAD7 nuclease on an ongoing basis or a product that physically contains the MAD7 nuclease. 

Inscripta is continuing its development of novel nucleic acid-guided endonucleases. Visit inscrip​ta​.com for future releases.

In addition to the MAD7 nuclease, we’re also engineering novel, bespoke enzymes for exclusive use by industry partners. Please contact us at info@​inscripta.​com for more information.