EnGen® SpRY Cas9

Catalog #	Concentration	Size	List Price	Quantity	Your Price
M0669M	20 µM	2500 pmol	$960.00		$864.00
M0669T	20 µM	500 pmol	$241.00		$216.90

Catalog #	Size	List Price	Your Price
M0669M	2500 pmol	$960.00	$864.00
M0669T	500 pmol	$241.00	$216.90

EnGen SpRY Cas9 from Streptococcus pyogenes is an engineered, RNA-guided, DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires a ~100 nucleotide single guide RNA (sgRNA) with complementarity to the 20-nucleotide region immediately upstream of a protospacer adjacent motif (PAM) on the dsDNA substrate. EnGen SpRY Cas9 encodes 11 point mutations (A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R) designed to diminish the requirement for a PAM. Unlike the canonical 5´-NGG-3´ PAM requirement of wild-type Spy Cas9, SpRY Cas9 has been demonstrated to have almost no PAM requirement in vitro, cleaving at many sites with a 5´-NNN-3´ PAM (although it exhibits a preference for 5´-NRN-3´ over 5´-NYN-3´ PAMs in vivo) ^(1,2). DNA cleavage by EnGen SpRY Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen SpRY Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the C-terminus of the protein.

M0669 Mechanism — EnGen SpRY Cas9 is a variant of Cas9 nuclease from S. pyogenes with several point mutations within the PAM interacting domain (1). Unlike wildtype Cas9, EnGen SpRY Cas9 is not constrained by the presence of an NGG PAM and can produce a double stranded break three nucleotides upstream of any trinucleotide sequence in in vitro applications.

M0669 Cleavage Specificty — Demonstration of EnGen® SpRY Cas9 cleavage flexibility. A) Schematic of pXba with restriction endonuclease BsrGI recognition sites denoted. The rectangular box contains the BsrGI recognition sequence with the cleavage sites denoted by red triangles. B) Sequences of three sgRNAs used to target BsrGI sites in pXba. Cleavage sites for both EnGen SpRY Cas9 and BsrGI are denoted by red triangles. SpRY Cas9 non-canonical PAMs are denoted in yellow text. C) 1 µg of pXba (22,563 bp) was digested with either 10 units of BsrGI or 1 µM EnGen SpRY Cas9 and 1 µM each of three sgRNAs targeting all three BsrGI sites present in pXba in 1X NEBuffer r3.1. All reactions were incubated at 37°C for 1 hour followed by 80°C for 5 minutes. The DNA banding pattern of the BsrGI and SpRY digests were compared by gel electrophoresis using the Agilent gDNA ScreenTape System on a 4200 TapeStation instrument. Cleavage of pXba by both BsrGI and SpRY Cas9 targeted to the BsrGI sites resulted in a nearly identical band pattern. Digestion of 1 µg of pXba with EnGen SpRY Cas9 and sgRNA concentrations as low as 50 nM resulted in a similar cleavage compared with 1 µM results. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. A vertical grey line marks where two different ScreenTapes were used.

M0669 Suitability for NEBuilder — Determination of suitability of EnGen SpRY Cas9 digestion reactions for subsequent use in NEBuilder HiFi DNA Assembly reactions. A) Schematic depicting the linearization and cloning strategy. 1 µg of pXba (22,563 bp) was linearized downstream of the start codon of the pVII orf with 50 nM EnGen SpRY Cas9 and 50 nM sgRNA (targeting sequence of 5´-CTTTTTGAGCAAACATGTCC-3´) in 1X NEBuffer r3.1 for 1 hour at 37°C. The reactions were either spin column purified or left unpurified before proceeding to DNA assembly. The NEBuilder HiFi DNA Assembly kit was used to insert an oligonucleotide encoding a nuclear localization signal (NLS) tag to pVII according to the recommended protocol. B) Inspection of purified and unpurified linearized pXba after EnGen SpRY Cas9 digestion using the Agilent gDNA ScreenTape System. Undigested pXba was run as a control, however circular DNA does not run accurate to size in the gDNA ScreenTape System. C) Measurement of the number of colonies grown after transformation with DNA assembly reactions using either purified or unpurified EnGen SpRY Cas9 digests. A no insert control was included to qualitatively assess how many transformants arise from undigested plasmid following EnGen SpRY Cas9 digest. Purification of linearized pXba prior to DNA assembly reduced the percentage of background colonies. 8-9 colonies of transformants derived from purified and unpurified EnGen SpRY Cas9 digests were picked and checked for proper DNA assembly by PCR using primers spanning the insert. 100 percent of clones from both purified and unpurified EnGen SpRY Cas9 digests displayed the correct insert (data not shown).

Product Source

An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes with C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

M0669T -20

	EnGen^® SpRY Cas9	M0669TVIAL	-20	1 x 500 pmol	20 µM
	NEBuffer™ r3.1	B6003SVIAL	-20	1 x 1.25 ml	10 X

M0669M -20

	EnGen^® SpRY Cas9	M0669MVIAL	-20	1 x 2,500 pmol	20 µM
	NEBuffer™ r3.1	B6003SVIAL	-20	1 x 1.25 ml	10 X

An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Notes

20 µM is equal to 3.25 mg/ml

References

Walton, et al. (2020). Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science. Apr 17;368(6488), 290-6. DOI: 10.1126/science.aba8853
Christie, et al. (2023). Precise DNA cleavage using CRISPR-SpRYgests. Nat. Biotechnol.. Mar 41, 409-16. DOI: 10.1038/s41587-022-01492-y

Protocols

In vitro digestion of plasmid DNA with EnGen SpRY Cas9 (NEB #M0669)

FAQs

Quality Control Assay

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.

Certificate of Analysis

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]

Safety Data Sheets

Legal And Disclaimer

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.

NEB Catalogue

Product Categories

Freezer Locations

Tools

Resources