Alt-R S.p Cas9 nuclease
Alt-R S.p. Cas9 Nuclease V3 is a high purity, recombinant S. pyogenes Cas9. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The S. pyogenes Cas9 enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease V3 enzyme with optimized Alt-R CRISPR gRNA in equimolar amounts.
Alt-R S.p. HiFi Cas9 nuclease
Alt-R S.p. HiFi Cas9 Nuclease V3 offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease V3 with the optimized Alt-R CRISPR-Cas9 gRNA (crRNA:tracrRNA) is highly recommended.
Alt-R S.p. Cas9 nickases
Cas9 nickases allow specific cutting of only one strand at the DNA target site. Cuts to both strands of DNA are accomplished by using either Alt-R S.p. Cas9 D10A Nickase V3 or Alt-R S.p. Cas9 H840A Nickase V3, with 2 gRNAs that target two neighboring Cas9 sites, one on either strand of the target region. This functionally increases the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the application note.
Alt-R S.p. dCas9 protein
Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA.
Alt-R A.s. Cas12a (Cpf1) nuclease
Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme is a high purity, recombinant Acidaminococcus sp. BV3L6 Cas12a. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The Cas12a enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme with optimized Alt-R CRISPR-Cas12a (Cpf1) crRNA in equimolar amounts.
Attention: Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cas12a nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cas12a. In addition, we suggest testing 3 or more crRNAs per target gene.
Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)
| ||Cas9 system||Cas12a system|
|Applications||General genome editing|
- For species with AT-rich genomes
- For regions with limiting design space for use of the CRISPR-Cas9 system
- gRNA options:
- crRNA and tracrRNA
- Cas9 endonuclease
- Nickases (H840A and D10A)
|Cas9 crRNA:tracrRNA (option 1)|
- Native: 42 nt
- Alt-R: 35–36 nt (36 nt recommended)
- Native: 89 nt
- Alt-R: 67 nt
|Cas9 sgRNA (option 2)|
- Alt-R: 99–100 nt (100 nt recommended)
- Native: 42–44 nt
- Alt-R: 40–44 nt (41 nt recommended)
- Class 2, Cas type II
- M.W.*: 162,200 g/mol
- Endonuclease domains: RuvC-like and HNH
- Class 2, Cas type V
- M.W.*: 156,400 g/mol
- Endonuclease domain: RuvC-like only
- Wild-type and HiFi: Blunt-ended cut 3 bases upstream of the protospacer sequence
- D10A nickase with paired gRNAs: 5′ overhang
- H840A nickase with paired gRNAs: 3′ overhang
- PAM site often destroyed during genome editing
- 5′ overhanging cut on the 5′ side of the protospacer sequence
- PAM site may be preserved after genome editing
|Current recommendations for Alt-R RNP delivery|
- Lipid-mediated transfection
- Electroporation (Alt-R enhancer recommended)
- Electroporation (Alt-R enhancer required)
* Molecular weight of Alt-R nuclease
† N = any base; V = A, C, or G
This comparison table is available for download (see page 2).