Alt-R™ CRISPR-Cpf1 System
Simple, 2-step delivery of ribonucleoprotein complexes (crRNA:Cpf1)
Figure 1. Overview of Alt-R™ CRISPR-Cpf1 System experiments for ribonucleoprotein (RNP) delivery by electroporation.
CRISPR-Cpf1 genome editing method uses the Cpf1 endonuclease to generate double-stranded breaks that contain a staggered 5′ overhang. Cpf1 only requires a single CRISPR RNA (crRNA) to specify the DNA target sequence (Figure 1). After cleavage, DNA is then repaired by non-homologous end-joining (NHEJ) or homology-directed recombination (HDR), resulting in a modified sequence. Alt-R CRISPR-Cpf1 reagents provide essential, optimized tools needed to use this pathway for genome editing research. A brief comparison of CRISPR-Cas9 and CRISPR-Cpf1 is provided at the end of this section.
|Alt-R™ CRISPR-Cpf1 System|
|Alt-R™ CRISPR-Cpf1 crRNA ||Target-specific RNA oligo, custom synthesized based on your sequence|
Alt-R™ A.s. Cpf1 Nuclease 2NLS ||• Protein that binds the Cpf1 crRNA, creating an experiment-ready, active ribonucleoprotein (RNP) complex |
• Contains 2 nuclear localization signals (NLSs) for optimal performance
|Alt-R™ Cpf1 Electroporation Enhancer||Cpf1-specific carrier DNA required for efficient electroporation of the RNP|
|Related reagents and kits|
|Cpf1 positive controls ||Order as custom crRNAs or oligos|
• HPRT crRNAs (human, mouse, or rat): To show that Cpf1 editing is occurring in your system during experimental optimization or troubleshooting
• HPRT PCR primers (human, mouse, or rat): To detect genome editing in experiments with HPRT crRNAs; for use with the Alt-R Genome Editing Detection Kit
Alt-R™ Genome Editing Detection Kit ||For mutation detection and estimating editing efficiency |
Components for genome editing
Cpf1 endonuclease from Acidaminococcus sp. BV3L6 along with a crRNA is capable of mediating genome editing in mammalian cells (Figure 2). The Alt-R CRISPR-Cpf1 System includes 3 main components: an optimized crRNA, A.s. Cpf1 endonuclease, and an electroporation enhancer. While electroporation of Cpf1 endonuclease as part of an RNP is the preferred method, the Alt-R CRISPR-Cpf1 crRNA is also compatible with A.s. Cpf1 from any source, including cells that stably express A.s. Cpf1 endonuclease.
- Alt-R CRISPR-Cpf1 crRNA and design tips
The Alt-R CRISPR-Cpf1 crRNA is a single, 40–44 base, guide RNA, comprised of a 20 base constant region (loop domain) and a 20–24 base target-specific region (protospacer domain). We typically recommend a 21 base protospacer domain for optimal activity. All Alt-R CRISPR-Cpf1 crRNAs are synthesized with proprietary chemical modifications, which protect the crRNA from degradation by cellular RNases and further improve on-target editing performance.
For crRNAs used with A.s. Cpf1, identify locations in your target region with the protospacer adjacent motif (PAM) sequence, TTTV, where V is A, C, or G. Your Alt-R CRISPR-Cpf1 crRNA will bind to the DNA strand opposite to the PAM sequence (Figure 2). Do not include the PAM sequence in your crRNA design. An example of a correct crRNA sequence is shown in Figure 3.
Once you enter your 20–24 base target sequence, 20 additional bases and the necessary modifications will automatically be added by the order entry system for a total of 40–44 RNA bases. These additional bases and modifications are necessary to create a complete Alt-R CRISPR-cpf1 crRNA. The system will also convert the final sequence to RNA—enter DNA bases only into the ordering tool (Figure 3).
Figure 3. How to enter your Cpf1 crRNA target sequence. Because the crRNA recognizes and binds 21 bases on the DNA strand opposite from the TTTV sequence of the PAM site, order your crRNA by entering the 20–24 bases downstream of the PAM site, in the forward orientation as shown. Enter only DNA bases into the order entry tool. If you are pasting your CRISPR target site from an online design tool, make sure you verify the correct strand orientation. Do not include the PAM site in your design. Common incorrect design examples are shown in red. PAM = protospacer adjacent motif; V = A, C, or G
- Alt-R A.s. Cpf1 Nuclease 2NLS
Alt-R A.s. Cpf1 Nuclease 2NLS enzyme is a high purity, recombinant Acidaminococcus sp. Cpf1. The enzyme includes 1 N-terminal nuclear localization sequence (NLS) and 1 C-terminal NLSs, as well as 3 N-terminal FLAG tags and a C-terminal 6-His tag. The Cpf1 enzyme must be combined with a crRNA to produce a functional, target-specific editing complex. For the best editing, combine Alt-R A.s. Cpf1 Nuclease 2NLS enzyme with optimized Alt-R CRISPR-Cpf1 crRNA in equimolar amounts.
In contrast to Streptococcus pyogenes Cas9, which recognizes an NGG PAM sequence, the A.s. Cpf1 PAM sequence is TTTV, which permits targeting of DNA sequences in AT-rich regions of the genome.
Alt-R™ A.s. Cpf1 Nuclease 2NLS
- Amount provided: 100 µg or 500 µg
- Molecular weight: 157,900 g/mol
- Concentration: 10 µg/µL in 50% glycerol, [63 µM]
- Endotoxin tested: <2 EU/mg
- Shipping conditions: dry ice
- Storage conditions: –20°C
- Alt-R Cpf1 Electroporation Enhancer
The Alt-R Cpf1 Electroporation Enhancer is a Cpf1-specific carrier DNA that is optimized to work with the Amaxa® Nucleofector® device (Lonza) and the Neon® Transfection System (Thermo Fisher) for increased transfection efficiency and therefore, increased genome editing efficiency.
Positive control crRNAs can be used to show that Cpf1 editing is occurring in your experiments, which can be useful when you are optimizing RNP delivery conditions or if you need to troubleshoot your experiments.
IDT scientists have designed and tested positive control crRNAs targeting HPRT. To order, copy and paste the appropriate sequence in the Cpf1 crRNA ordering page:
Human HPRT1, Cpf1 Positive Control crRNA: GGTTAAAGATGGTTAAATGAT
Mouse Hprt, Cpf1 Positive Control crRNA: GGATGTTAAGAGTCCCTATCT
Rat Hprt1, Cpf1 Positive Control crRNA: ACCGCCCCCCCCATACCCCAA
Attention: Unlike S. pyogenes Cas9, which cleaves most potential NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cpf1 nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cpf1. In addition, we suggest testing 3 or more crRNAs per target gene.
IDT scientists have also designed and tested PCR primers (Alt-R HPRT PCR Primer Mixes for human, mouse, or rat) for use with the Alt-R Genome Editing Detection Kit to detect editing or estimate editing efficiency in samples transfected with the positive control HPRT crRNAs.
Negative control crRNAs are important for showing that transfection of the RNP complex is not responsible for observed phenotypes. Amplification of DNA from these negative control samples with your experimental primers and cycling conditions should result in only full-length products with the Alt-R Genome Editing Detection Kit (i.e., in a T7EI assay). Note, that this result does not rule out off-target effects of your experimental crRNA.
IDT scientists have computationally designed and tested negative control crRNAs to be non-targeting in human, mouse, and rat genomes. To order, copy and paste the appropriate sequence in the Cpf1 crRNA ordering page:
Cpf1 Negative Control crRNA #1: CGTTAATCGCGTATAATACGG
Cpf1 Negative Control crRNA #2: CATATTGCGCGTATAGTCGCG
Cpf1 Negative Control crRNA #3: GGCGCGTATAGTCGCGCGTAT
Comparison of CRISPR genome editing using Cas9 vs. Cpf1
||General genome editing
||• For species with AT-rich genomes
• For regions with limiting design space for use of the CRISPR-Cas9 system
• Cas9 endonuclease
• Cpf1 endonuclease
||• Native: 42 nt
• Alt-R: 35–36 nt (36 nt recommended)
|• Native: 42–44 nt
• Alt-R: 40–44 nt (41 nt recommended)
||• Native: 89 nt
• Alt-R: 67 nt
| (not applicable)
||• Class 2, Cas type II
• M.W.*: 163,700 g/mol
• Endonuclease domains: RuvC-like and HNH
|• Class 2, Cas type V
• M.W.*: 157,900 g/mol
• Endonuclease domain: RuvC-like only
|Double-stranded DNA cleavage
||• Blunt ended cut 3 bases upstream of the protospacer sequence
• 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
|• Electroporation with Alt-R™ enhancer
* Molecular weight of Alt-R™ nuclease
† N = any base; V = A, C, or G