MB-CasFire is a thermostable Type V CRISPR nuclease guided by a single guide RNA (gRNA). The broad activity range (37-65°C) and robust trans-nuclease activity of MB-CasFire enable target detection in OnePot DETECTR® assays when coupled with isothermal amplification methods, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA).
Maximize assay flexibility with compatibility with LAMP/RT-LAMP and RPA/RT-RPA workflows, including Bst-XT WarmStart™ Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1712)
Enable the detection of single nucleotide polymorphisms (SNPs)
Support flexible targeting with a YYN PAM sequence
Confirm that amplification produced the intended sequence with guide-directed target recognition
Supports isothermal workflows with robust activity from 37-65°C.
MB-CasFire is thermostable Type V CRISPR nuclease guided by a single guide RNA (gRNA). The broad activity range (37-65°C) and robust trans-nuclease activity of MB-CasFire enable target detection in OnePot DETECTR® assays when coupled with isothermal amplification methods, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). MB-CasFire is programmed by a 40-nt guide RNA (gRNA). Targeting requires a gRNA complementary to the target site as well as a 5’ YYN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence.
Figure 1: MB-CasFire Workflow
Figure 2: OnePot DETECTR LAMP assay using MB-CasFire detects the C-to-T SNP (S91F) in gyrA, a ciprofloxacin-resistance marker in Neisseria gonorrhoeae
A. gBlocks representing the wild-type (WT) sequence and a mutant (MUT) version carrying the C-to-T change that converts serine (S) to phenylalanine (F) were used as templates. LAMP primers designed using NEB LAMP Primer tool and WT- or MUT-specific guide RNAs (gRNAs) were designed to target the substitution site.
B. OnePot DETECTR reactions (2,000 copies/µl and NTCs, in triplicate) were assembled following the Bst-XT WS LAMP protocol. The reactions were run at 64°C for 45 minutes in a real-time thermocycler (Bio-Rad® CFX Opus 96). LAMP amplification was monitored using NEB #B1700 dsDNA intercalating dye in the SYBR/FAM channel, and the MB-CasFire signal from the Alexa 594-labeled Cas reporter was read in the Texas Red® channel.
The data shows the LAMP signal alone did not distinguish WT from MUT templates. In contrast, target-specific gRNA-MB-CasFire complexes discriminated the two templates, showing that MB-CasFire resolves targets at single-nucleotide resolution.
Figure 3: MB-CasFire eliminates nonspecific amplification in a OnePot DETECTR RT-RPA assay OnePot DETECTR RT-RPA reactions were assembled using T4 UvsX/UvsY/gp32 proteins, Bsu DNA Polymerase Large Fragment, ProtoScript® II Reverse Transcriptase, RNase H and MB-CasFire.
RT-RPA primers for the SARS-CoV-2 N gene were tested along with a gRNA designed within the target amplicon region. Every condition was run in triplicate at 500 copies/µl, using Twist Control 2 RNA as template and Jurkat RNA in the NTCs. Reactions were incubated at 42°C for 60 minutes in a real-time thermocycler (Bio-Rad CFX Opus 96). RT-RPA amplification was monitored using NEB #B1700 dsDNA intercalating dye in the SYBR/FAM channel, and MB-CasFire signal from the Alexa 594-labeled Cas reporter was read in the Texas Red channel.
Intercalating dye-based RT-RPA alone produced significant non-specific amplification, whereas the MB-CasFire-gRNA complex gave a specific signal. These results indicate that coupling RPA with MB-CasFire and a target-specific gRNA can improve specificity and enable direct detection of the target amplicon.
This product is related to the following categories:
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Ineligible item added to cart
Based on your Freezer Program type, you are trying to add a product to your cart that is either not allowed or not allowed with the existing contents of your cart. Please review and update your order accordingly. If you have any questions, please contact Customer Support at freezers@neb.com or 1-800-632-5227 x 8.
Choose your country
North America
Europe
Asia-Pacific
Session Expired
Your session has expired
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Please sign in to continue shopping.
Institution Changed
Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.
Sign in to your NEB account
To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.
Powered by Bioz
