CRISPR-Cas12a genome editing method uses the Cas12a endonuclease to generate double-stranded breaks that contain a staggered 5′ overhang. Cas12a 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-Cas12a reagents provide essential, optimized tools needed to use this pathway for genome editing research. A brief comparison of CRISPR-Cas9 and CRISPR-Cas12a is provided at the end of this section.
|Alt-R CRISPR-Cas12a System|
|Alt-R CRISPR-Cas12a (Cpf1) crRNA||Target-specific RNA oligo, custom synthesized based on your sequence|
|Alt-R A.s. Cas12a (Cpf1) Nuclease V3|
- Protein that binds the Cas12a crRNA, creating an experiment-ready, active ribonucleoprotein (RNP) complex
- Contains nuclear localization signals (NLSs) for optimal performance
|Alt-R Cas12a (Cpf1) Electroporation Enhancer||Cas12a-specific carrier DNA required for efficient electroporation of the RNP|
|Related reagents and kits|
|Alt-R HDR Enhancer||For improved rates of homology-directed repair|
|Cas12a positive controls||Order as custom crRNAs or oligos|
- HPRT crRNAs (human, mouse, or rat): To show that Cas12a 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
Cas12a endonuclease from Acidaminococcus sp. BV3L6 along with a crRNA is capable of mediating genome editing in mammalian cells (Figure 2). The Alt-R CRISPR-Cas12a System includes 3 main components: an optimized crRNA, A.s. Cas12a endonuclease, and an electroporation enhancer. While electroporation of Cas12a endonuclease as part of an RNP is the preferred method, the Alt-R CRISPR-Cas12a crRNA is also compatible with A.s. Cas12a from any source, including cells that stably express A.s. Cas12a endonuclease.
Alt-R CRISPR-Cas12a (Cpf1) crRNA and design tips
The Alt-R CRISPR-Cas12a (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-Cas12a (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. Cas12a,
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-Cas12a (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.
After 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-Cas12a (Cpf1) crRNA. The system will also convert the final sequence to RNA—enter DNA bases
only into the ordering tool (Figure 3).
Alt-R A.s. Cas12a (Cpf1) nuclease
Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme is a high purity, recombinant Acidaminococcus sp. Cas12a. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The Cas12a enzyme must be combined with a crRNA 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.
In contrast to Streptococcus pyogenes Cas9, which recognizes an NGG PAM sequence, the A.s. Cas12a PAM sequence is TTTV, which permits targeting of DNA sequences in AT-rich regions of the genome.
Alt-R Cas12a (Cpf1) Electroporation Enhancer
The Alt-R Cas12a (Cpf1) Electroporation Enhancer is a Cas12a-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.Alt-R CRISPR-Cas9 Control crRNAs and PCR Assays
Alt-R HDR Enhancer
Alt-R HDR Enhancer is a small molecule compound that increases homology-directed repair. Alt-R HDR Enhancer exhibits its activity in multiple cell lines, including both adherent and suspension cell lines. Its activity is independent of the enzyme employed;
for example, it can be used either with Alt-R S.p. Cas9 Nuclease V3 or Alr-R A.s. Cas12a (Cpf1) Nuclease V3. This versatile reagent is also compatible with electroporation and lipofection methods.
Positive control crRNAs can be used to show that Cas12a 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.
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. 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.
Our scientists have designed and tested positive control crRNAs targeting HPRT (Figure 4). To order, copy and paste the appropriate
sequence in the Cpf1 crRNA ordering page:
Human HPRT1, Cas12a Positive Control crRNA: GGTTAAAGATGGTTAAATGAT
Mouse Hprt, Cas12a Positive Control crRNA: GGATGTTAAGAGTCCCTATCT
Rat Hprt1, Cas12a Positive Control crRNA: ACCGCCCCCCCCATACCCCAA
Our 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.
Our 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 Cas12a (Cpf1) crRNA ordering page:
Cas12a Negative Control crRNA #1: CGTTAATCGCGTATAATACGG
Cas12a Negative Control crRNA #2: CATATTGCGCGTATAGTCGCG
Cas12a Negative Control crRNA #3: GGCGCGTATAGTCGCGCGTAT
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).