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Which Cas9 enzyme to use
Comparison of Alt-R Cas9 Nucleases and Nickases. Click here to download PDF
Alt-R S.p. Cas9 nuclease
Alt-R S.p. Cas9 Nuclease V3 is the standard Cas9 used for general genome editing. It 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 Alt-R CRISPR gRNA in equimolar amounts.
Alt-R S.p. HiFi Cas9 nuclease
Alt-R S.p. HiFi Cas9 Nuclease V3 is also used for general genome editing, but it 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-GFP (or RFP) nuclease
The Alt-R S.p. Cas9-GFP V3 and S.p. Cas9-RFP V3 nucleases are high purity, recombinant S. pyogenes Cas9 enzymes that are expressed as fusion proteins with nuclear localization sequences (NLSs) and C-terminal 6-His tags. These
enzymes have on-target functionality comparable to wild-type S.p. Cas9 and are designed for applications that require post‑transfection visualization of the protein or enrichment of edited cells using fluorescence-activated cell sorting (FACS).
These enzymes should be combined with Alt-R CRISPR gRNA in equimolar amounts.
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 two gRNAs that target
two neighboring Cas9 sites, one on either strand of the target region. There are two main reasons to consider using nickases. First, the use of two neighboring gRNAs instead of one gRNA (as used with Alt-R S.p. Cas9) can decrease off-target
effects. Second, the rate of HDR is increased. 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. The primary use of
dCas9 protein is to block transcription at a specific site on the genome. This is known as CRISPRi and is an alternative to RNAi for knockdown instead of knockout of genes.
Like the other Alt-R enzymes, Alt-R S.p. dCas9 Protein V3 is provided as 10 mg/mL in 50% glycerol, and it can be diluted in PBS or Opti-MEM media before use.
Cas12a (Cpf1) proteins
Alt-R A.s. Cas12a (Cpf1) V3 nuclease
Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme is a high purity, recombinant Acidaminococcus sp. BV3L6 Cas12a. It is useful for targeting AT-rich regions when the Cas9-specific PAM sequence (NGG) is not available. 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.
Alt-R A.s. or L.b. Cas12a (Cpf1) Ultra Nucleases
The Alt-R Cas12a (Cpf1) Ultra Nucleases are also useful for targeting AT-rich regions without available Cas9-specific PAM sequences. However, they have much higher on-target potency than wild-type A.s. Cas12a (Cpf1). The Alt-R
Cas12a (Cpf1) Ultra also can recognize many TTTT PAM sites in addition to TTTV motifs, increasing target range for genome editing studies. Furthermore, the new Alt-R Cas12a (Cpf1) Ultra nucleases are active at room temperature,
making them flexible tools for applications requiring delivery at lower temperatures.
Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)
General genome editing
For species with AT-rich genomes
For regions with limiting design space for use of the CRISPR-Cas9 system
crRNA and tracrRNA
Nickases (H840A and D10A)
Cas9-GFP (or RFP)
Ultra (improved performance)
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
TTTV for Cas12a V3
TTTN for Cas12a Ultra
Current recommendations for Alt-R RNP delivery
Electroporation (Alt-R enhancer recommended)
Electroporation (Alt-R enhancer recommended)
* Molecular weight of Alt-R nuclease † N = any base; V = A, C, or G
Improved editing efficiency using Alt-R S.p. Cas9 Nuclease V3
Alt-R S.p. Cas9 Nuclease V3 is designed to maximize the efficiency of genome editing across a broad number of sites. Modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced cleavage, particularly
at difficult targets (Figure 1).
Figure 1. Alt-R S.p. Cas9 Nuclease V3 genome editing efficiency even at challenging target sites. Ribonucleoprotein (RNP) complexes were formed with 1 of the 2 wild-type Cas9 proteins—Alt-R S.p. Cas9 Nuclease 3NLS (light blue) or Alt-R S.p. Cas9 Nuclease V3 (dark blue), combined with an Alt-R crRNA:tracrRNA complex targeting one of 11 loci on the human HPRT gene. RNP complexes (4 µM) were delivered into HEK-293 cells by nucleofection. Total editing at the on-target loci was calculated by NGS. n = 1.
Increased specificity using Alt-R S.p. HiFi Cas9 Nuclease V3
As with the wild-type Alt-R Cas9 Nuclease V3, modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced on-target cleavage by Alt-R S.p. HiFi Cas9 Nuclease V3. However, Alt-R HiFi Cas9 Nuclease V3 also provides
superior cutting specificity (minimized off-target editing; Figure 2).
Figure 2. Alt-R S.p. HiFi Cas9 Nuclease V3 facilitates near-WT on‑target editing potency and reduces off-target site editing. RNP complexes were formed with either Alt-R S.p. Cas9 Nuclease V3 or Alt-R S.p. HiFi
Cas9 Nuclease V3, combined with an Alt-R crRNA:tracrRNA complex targeting the EMX1 gene. RNP complexes (4 µM) were delivered into HEK-293 cells via nucleofection. Indel formation at the on-target locus as well as nine known off-target sites
were measured by NGS (indicated on the y axis in log scale). n = 1.
Potent editing with the Alt-R S.p. Cas9 nucleases
The Alt-R CRISPR-Cas9 System includes potent Alt-R S.p. Cas9 nucleases. When Alt-R S.p. Cas9 Nuclease 3NLS was combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperformed other editing approaches (Figure 3). You can expect even better editing efficiency with Alt-R S.p. Cas9 Nuclease V3 (see Figure 2). RNP transfections also provide control of amount of editing complexes used, and the non-renewable Cas9 RNP is cleared after a short duration by endogenous mechanisms, limiting off-target editing.
Figure 3. Experiment showing that lipofection of Alt-R CRISPR‑Cas9 System components as a ribonucleoprotein outperforms other transient CRISPR-Cas9 approaches. Alt-R CRISPR HPRT Control crRNA complexes for human, mouse, or rat were complexed
with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as part of a Cas9 RNP (containing Alt-R S.p. Cas9 Nuclease 3NLS, pre-complexed with the crRNA and tracrRNA) into human (HEK-293),
mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression approaches, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9. Error bars represent SD, n = 3.
Figure 4. IDT fluorescent CRISPR proteins maintain consistent on-target activity across multiple guides. Alt-R CRISPR-Cas9 sgRNAs were designed to target NGG PAM sites within the human HPRT gene. Guides were complexed with Alt-R S.p. Cas9 Nuclease V3, Cas9-GFP, or Cas9-RFP to form RNPs. RNPs were then delivered into HEK293 cells using the Lonza 96 well shuttle nucleofector at a concentration of 2.0 µM. After 48 hrs, genomic DNA was isolated (QuickExtract™ solution,
Epicenter), and editing was assessed by T7El mismatch endonuclease assay. Error bars represent SD, n = 3.
As shown in Figure 5, Alt-R fluorescent CRISPR nucleases enable enrichment of edited cells by fluorescent activated cell sorting (FACS).
Figure 5. Fluorescent CRISPR proteins can be used to enrich for edited cells by fluorescence activated cell sorting. (A) RNPs consisting of either Cas9-GFP or Cas9 V3 complexed with CRISPR‑Cas9 sgRNAs targeting two sites
in the HPRT gene were delivered into HEK293 cells using either Lipofectamine™ RNAiMAX (Thermo Fisher) at 10 nM RNP or using a Nucleofector™ system (Lonza) at 2 µM RNP. The graphs show the GFP signal versus cell count, where cell
count has been normalized to the mode for cells that had either Cas9-GFP or Cas9 V3 delivered using either lipofection or Nucleofection™. (B) Alt-R CRISPR-Cas9 sgRNAs were designed to target NGG PAM sites throughout the human
genome. Guides were complexed with either Cas9-GFP or Alt-R Cas9 V3. RNPs were delivered into HEK293 cells using Lipofectamine™ RNAiMAX at 10 nM final concentration. After ~18 hrs, cells were sorted using a FACSAria™ II (Becton Dickinson)
cell sorter into three subpopulations, GFP High: top 20%, Medium: 80–60%, and Low: Bottom 60% of cells based on GFP signal. Cells were then replated, and genomic DNA was isolated after 48-72 hrs. Editing was analyzed by NGS, n =
1. (C) Confocal images of HEK293 cells taken approximately 18 hours after delivery of either Cas9-GFP or Alt-R Cas9 V3 protein complexed with Alt-R CRISPR-Cas9 sgRNA delivered by Nucleofection™ at 2 µM RNP. Prior to imaging,
live cells were incubated with Hoechst 33342 and washed with PBS. Cells were imaged in complete media in a chambered coverglass using a Leica SP8 confocal microscope.
To enhance activity, we introduced multiple modifications to the Cas12a protein that support notable improvement in overall editing efficiency. The new Alt-R Cas12a (Cpf1) Ultra nuclease has higher on-target potency than the wild-type A.s. Cas12a (Cpf1). The new Alt-R Cas12a (Cpf1) Ultra also can recognize many TTTT PAM sites in addition to TTTV motifs, increasing target range for genome editing studies (Figure 6). Furthermore, the new Alt-R Cas12a (Cpf1) Ultra nuclease
is active at room temperature, making it a flexible tool for applications requiring delivery at lower temperatures.
Figure 6. New A.s. Cas12a (Cpf1) Ultra exhibits increased genomic editing efficiency in Jurkat and HEK-293 cells. Ribonucleoprotein (RNP) complexes were formed with wild type (WT) or Alt-R A.s. Cas12a (Cpf1) Ultra (Ultra), combined with crRNAs synthesized for 120 genomic loci to be delivered in Jurkat cells and 96 genomic loci to be delivered in HEK-293 cells. RNP complexes (4 μM) were delivered into Jurkat and HEK-293 cells via a Nucleofector™ system (Lonza) in the presence of Alt-R Cas12a (Cpf1) Electroporation Enhancer. Genome editing efficiencies were determined by target amplification followed by next generation sequencing on an Illumina instrument. The Cas12a-associated PAM sequences are indicated below the graph. n = 426, with 213 data points for WT and 213 data points for Cas12a Ultra. Each dot represents a single sample.
The electroporation enhancer is recommended for efficient genome editing with the CRISPR-Cas12a (Cpf1) system
The Alt-R Cas12a (Cpf1) Electroporation Enhancer is a Cas12a-specific carrier DNA that is optimized to work with the Nucleofector™ device (Lonza) and the Neon™ Transfection System (Thermo Fisher) for increased transfection efficiency and therefore, increased genome editing efficiency (Figure 7). The electroporation enhancer is non-targeting and shows no integration into the target site based on next-generation sequencing experiments.
Figure 7. Alt-R Cas12a (Cpf1) Electroporation Enhancer is required for efficient CRISPR editing in ribonucleoprotein (RNP) electroporation experiments. HEK-293 cells were electroporated with 5 µM RNP (Alt-R A.s. Cpf1 Nuclease
2 NLS complexed with Alt-R CRISPR-Cas12a (Cpf1) crRNA) as instructed in the Alt-R CRISPR-Cas12a (Cpf1) User Guide—RNP electroporation, Nucleofector™ system (available at www.idtdna.com/CRISPR-Cpf1). Twelve Cas12a PAM sites in the HPRT gene were targeted by Alt-R CRISPR-Cas12a (Cpf1) crRNAs. The electroporation reactions contained either
no (dark blue) or 3 µM (light blue) Alt-R Cas12a (Cpf1) Electroporation Enhancer. Editing efficiency (n =3) was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required
for T7EI endonuclease assays. PAM = protospacer adjacent motif (Cas12a PAM sequence is TTTV); x-axis: numbers specify gene locations; S = sense strand; AS = antisense strand.
User guides and protocols
Improved enzymes: All Alt-R enzymes (Cas9 nuclease, HiFi Cas9 nuclease, Cas9 nickases, and Cas12a (Cpf1) nuclease) have recently been further optimized to deliver the most trusted results in your research. The latest versions (Alt-R S.p. Cas9 Nuclease V3 and A.s. Cas12a (Cpf1) Ultra) can be directly substituted into the protocols in place of the prior Alt-R enzymes.
Depending on your method of assessment, your editing efficiency may be underrepresented. Mismatch endonucleases may under-estimate actual editing compared to direct sequencing, as T7EI does not detect single base changes.
In addition, not every sequence associated with a PAM site performs the same. For example, polymorphisms in the protospacer binding site may reduce editing efficiency. Base mismatches also become more detrimental to editing the closer they are to the PAM site.
We recommend that you try 2 or 3 different PAM sites in your gene of interest to identify a site that provides optimal editing efficiency. Also, be sure to include control experiments. Alt-R™ CRISPR-Cas9 HPRT Positive Controls and Alt-R™ CRISPR-Cas9 Negative Controls are available for human, mouse, and rat.
Please feel free to contact us if you need additional assistance; having the results of your control experiments available will facilitate our ability to help you. For more information, go to www.idtdna.com/ContactUs.
DNA with homology to the sequences flanking a double-stranded break (DSB) can serve as template for error-free homology directed-repair (HDR) of the DSB. The efficiency of HDR is determined by the concentration of donor DNA present at the time of repair, length of the homology arms, cell cycle, and activity of the endogenous repair systems in the particular cell . These factors contribute to the high variability of HDR efficiency observed across different cell lines, and particularly in immortalized cells . Typically, in replicating mammalian cells, donor arms are at least 500 bp in length . However, it is important to determine the optimal HDR conditions for your cell line.
Inserts between the homology arms are frequently in the 1–2 kb range . While longer inserts are possible, the efficiency of recombination decreases as the insert size increases . Finding successfully integrated inserts is likely to be challenging when inserts are greater than 3 kb in most mammalian cells.
Single-stranded oligo DNA (ssODN) has recently been identified as a substrate that is preferred by the HDR mechanism and often achieves good efficiency with homology arms as short as 40 bp [6,7]. The drawback to using ssODNs is that they are limited in length to a few hundred bases, so the insert size is limited. When using Alt-R HDR Donor Oligos as templates for a short insertion, tag, or SNP conversion, we have found arm lengths of 30–60 nt to be sufficient. Modified, linear double-stranded DNA (dsDNA) can also be used as a donor template. For longer, modified dsDNA donors (Alt-R HDR Donor Blocks), we have found homology arm lengths of 200–300 bp to be sufficient for HDR.
For HDR designs, refer to our HDR Design Tool which incorporates these recommendations.
Yes. The delivery of the CRISPR-Cas9 ribonucleoprotein (RNP) complexes with Alt-R Cas9 nickases is similar to RNP with the standard Alt-R S.p. Cas9 Nuclease V3, so the same protocol can be used with these enzymes. However, with the Cas9 nickases, two guide RNAs are required to generate a double-strand break, while only one guide RNA is required for the standard nuclease.
When preparing materials to deliver two guide RNAs simultaneously, first prepare RNPs with each guide RNA separately before RNP delivery.
We have successfully used electroporation in internal experiments to deliver Cas12a RNP complexes to cells in culture. In many cell lines, Alt-R™ Cas12a Electroporation Enhancer, a non-targeting carrier DNA, is also required for efficient delivery of the RNP complex. IDT protocols using Cas12a and electroporation are included here, and these Cas12a DECODED articles include functional data and details:
The PAM sequence for the Cas12a (Cpf1) system is TTTV , where "V" is a A, C, or G. The Cas12a PAM sequence can be advantageous when working with T-rich target sequences. In contrast, the Cas9 PAM sequence is NGG.
The Alt-R Cas9 nucleases and nickases are supplied at concentration of 10 mg/mL in 25 mM Tris-Cl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol (v/v), pH 7.4, with the exception of the Alt-R™ S.p. Cas9 V3, glycerol-free. The Alt-R™ S.p. Cas9 V3, glycerol-free is provided at a concentration of 10 mg/mL in a proprietary buffer without glycerol.
It depends upon the experimental design. Cas9 D10A will cut the target strand of DNA (which does not contain the PAM sequence), while Cas9 H840A will cut the non-target strand (which contains the PAM sequence). The availability and location of PAM sites within the target region of DNA needs to be assessed.
No. The PAM sequence is located on the non-complementary strand. That is, it is on the strand of DNA that contains the same DNA sequence as the target crRNA . The PAM sequence should not be included in the design of the crRNA.
The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.