Cas9 vs. Cas12a
Last update: Sep 2020
Which one works better-CRISPR-Cas9 or CRISPR-Cas12a?
The table below describes the differences between systems employing Cas9 and Cas12a, providing a general comparison between the characteristics and performance of the two nucleases. This brings together the knowledge from previous sections within CRISPR-Cas Technologies topic. If you would like to revise the basics of the CRISPR-Cas Systems before reading this section, click on the button to the right (bottom if on mobile).
Table 1. Comparison between CRISPR-Cas9 and CRISPR-Cas12a
CRISPR-Cas9 and CRISPR-Cas12a in Microorganisms
CRISPR Cas systems are widely used in industrial microbiology for the production of biofuels, pharmaceutical compounds, et cetera. Thanks to genetically engineered microorganisms, production has become most cost-effective, and yields were increased. CRISPR-Cas9 and CRISPR-Cas12a enable the creation of ever-improving microbial cell factories. Multiplexed genome editing proved to be especially useful in this context when re-engineering both bacterial and eukaryotic cells. CRISPR-Cas systems are used particularly often to edit yeast and filamentous fungi.
Cas9 isolated from Streptococcus pyogenes (SpCas9) is most commonly used to introduce genetic edits to microorganisms. Several variants of Cas12a are employed less frequently. The 4 types of the most popular Cas nucleases are described in Table 2.
Both dCas9 and dCas12a are used in CRISPRi and CRISPRa in microorganisms. In the case of CRISPRi in eukaryotic microorganisms, dCas nucleases are fused to mammalian transcriptional repressor domain Mxi1 or the KRAB domain. In bacteria, dCas nuclease alone is often sufficient to block transcription. In the case of CRISPRa in eukaryotic microorganisms, P16, VP64, Gal4AD, or VPR activator domains are fused to dCas nucleases. In bacteria, the omega (ω) subunit of the RNA polymerase is commonly employed.
Table 2. Cas nucleases used for genome editing
Multiplex editing using CRISPR-Cas systems allows minute effective alterations to the microbial metabolic networks. CRISPR-Cas9 is still the preferred tool for this procedure. As we can see in Table 3, the editing efficiency of genome editing varies greatly depending on which microorganism and which Cas nuclease we use and what type and number of edits we want to make. The results are slightly inconclusive when it comes to determining whether Cas9 or Cas12a is more effective.
Table 3. Multiplexed genome editing outcomes in industrially relevant microorganisms
As a principle, Cas9 is used more frequently than Cas12a, which reduces the amount of available data on the latter. The general pattern for both Cas9 and Cas12a is that the editing efficiency decreases with the number of gene edits, regardless of the type of edit. For example, editing efficiency of generating knockouts in Escherichia coli [MG1655] using SpCas9 was 97% ± 4% for 2 knockouts, but only 47% ± 8% for 3 knockouts. FnCas12a (FnCpf1) generated 2 knockouts in Saccharomyces cerevisiae[CEN.PK113–5D (n)] with 100% efficiency, but for 4 simultaneous knockouts, the efficiency dropped to 85%.
The comparison between Cas9 and Cas12a is indeed difficult, as the studies mentioned in Table 3 rarely used the same microorganism strain and the same type of edit, for example, while the researchers attempted to edit the genome of Escherichia coli[MG1655] and Streptomyces coelicolor[M145] using both SpCas9 and FnCas12a, the types of modifications differed. All studies on eukaryotes shown in Table 3 were performed on Saccharomyces cerevisiae, which could make the results slightly easier to compare, but unfortunately, the strains used vary. SpCas9 used to alter Saccharomyces cerevisiae[CEN.PK2–1c (n)] exhibited 64% efficiency when generating 3 knockouts, while FnCpf1 showed 100% efficiency for 2, and 85% efficiency for 4 knockouts in Saccharomyces cerevisiae[CEN.PK113–5D (n)].
Figure 1 highlights the large research gap between Cas9 and Cas12a and justifies the difficulty in drawing definite conclusions about the usefulness of Cas12a compared to Cas9, given the insufficient number of studies investigating Cas12a. This could be because Cas9 was discovered several years prior to Cas12a, gaining immediate popularity among the scientists. As the number of publications involving Cas12a increases, more should be known about its potential.
CRISPR-Cas9 and CRISPR-Cas12a in plants
So far, CRISPR-Cas systems were used to edit various crucial crops, such as rice, corn, wheat, soybean, and tomato. In contrast to microorganisms, there is much more information available on the efficiency of genome editing using Cas9 and Cas12 in plants.
Banakar et al. based in the USA compared the CRISPR-Cas9 and Cas12a Ribonucleoprotein Complexes from the perspective of genome editing efficiency in Rice Phytoene Desaturase (OsPDS) Gene. Delivering the CRISPR reagents into plant cells as ribonucleoprotein (RNP, gRNA, and Cas nuclease complex) complexes allows transient genome editing, bypassing the usual procedure of cloning and vector construction altogether. The nucleases tested were WT Cas9, High Fidelity (HiFi) SpCas9, SpCas9 D10A (nickase), AsCas12a (Acidaminococcus sp. BV3L6) and LbCas12a (Lachnospiraceae bacterium ND2006). The mutagenesis of PDS was successful for WT Cas9, and HiFi Cas9 and LbCas12a only. Of those three, LbCas12a had the highest editing efficiency. Moreover, while using Cas9 nucleases resulted in 1-2 bp indels and 20-30 bp deletions and PMA site loss, LbCas12a produced 2-20 bp deletions and preserved the PAM site. LbCas12a exhibits improved function at lower temperatures, which is optimal for use in ectothermic organisms, e.g., zebrafish and plants
Banakar et al. decided to deliver Cas nucleases and gRNAs in their experiments so that the CRISPR-Cas system is only active transiently, which reduced the probability of off-target editing. Phytoene desaturase (PDS) is an enzyme assisting in the conversion of phytoene into zeta carotene in the carotenoid biosynthetic pathway. It is encoded by a single copy gene in rice. Knocking out PDS causes albino phenotype in callus tissue or in plant leaves, which makes the phenotype screening easy.
In the experiment, all nucleases were targeting the same DNA sequence. Cas9 nucleases used 36 nt crRNA containing 20 nt of targeting sequence, and Cas12a nucleases used 41 nt crRNA that containing 21 nt of targeting sequence. As shown in Table 4, the GC content of all crRNAs was similar.
Table 4. Cas nucleases and crRNAs used in the experiment
Cas nucleases and their gRNAs were delivered as RNP complexes into 5-day-old mature seed-derived rice embryos, along with a pCAMBIA1301 plasmid used for screening. For each type of RNP complex, 60 embryos were bombarded and then cultured on a medium containing hygromycin. Hygromycin-resistant calli were then subjected to next-generation sequencing (NGS) analysis. The results are summarized in Table 5.
Table 5. Transformation frequencies and editing efficiency of five CRISPR-Cas models
While the transformation frequency of LbCas12a was lower than that of WT Cas9 and HiFi Cas9, its editing efficiency was the highest at 32.3%. This shows that for some Cas variants and experimental designs in certain plants, CRISPR-Cas12a can exhibit higher genome editing efficiency than CRISPR-Cas9. Moreover, since the PAM sequence is preserved after editing with CRISPR-Cas12a, it is possible to target the same genomic locus again in the future. CRISPR-Cas12a could improve the frequency of HDR over NHEJ. Additionally, Cas12a could target and cleave the same sequence repeatedly.
The high editing efficiency of LbCas12a compared to AsCas12a in experiments involving the delivery of RNP complexes has been demonstrated not only in rice but also in soybean and tobacco protoplasts. LbCas12a also scores better when Cas nucleases are introduced as plasmid molecules into rice, Arabidopsis, and corn protoplasts. Cas12a nucleases are generally more active at higher temperatures of around 37°C, which is especially useful for human genome editing. However, the temperature in the transformation experiments performed by Banakar et al. was 28°C, which probably reduced the activity of both LbCas12a and AsCas12a.
Apart from the research conducted by Banakar et al., there are many more investigations using CRISPR-Cas12a to edit plant genomes. Those experiments are summarized in Table 6. Another experiment comparing Cas9 and Cas12a efficiency was performed by Yin et al., who used both nucleases to knock out the Epidermal Patterning Factor like-9 (EPFL9) gene. Again, the system employing LbCas12b produced a higher ratio of mutated T0 plants. Also, LbCpf1 demonstrated a maximum deletion size of 63 bp, while Cas9 showed a maximum deletion size of 37 bp. Endo et al. achieved 28.2% mutation frequencies in tobacco and 47.2% in rice using CRISPR-Cas12a.
Table 6. Recent work involving CRISPR-Cas12a systems
Since the sequence encoding Cas12a is shorter than that of Cas9, the size of the plant transformation vector can be reduced. CRISPR construct containing Cas 12a can be delivered into plant cells using Agrobacterium, bombardment, and PEG. PEG-mediated protoplast transformation allows DNA-free or vector-less editing in plants as well as mammalian cells. DNA-free edit (without T-DNA integration) has been already made in soybean and wild tobacco protoplasts.
Gene insertion using CRISPR-Cas9 usually has a 2.5–4.1% insertion rate for maize and 2.0–2.2% in rice. Interestingly, Begemann et al. achieved up to 8% insertion rates when FnCas12a and LbCas12a were employed in the CRISPR-Cas system.
One limitation of Cas12a is the potential formation of secondary structures in its crRNA, which is shorter than that of Cas9. This results in decreased editing efficiency. An example of this is an experiment targeting the glossy2 gene in maize by SpCas9 and LbCpf1 in Agrobacterium-mediated genome editing. T0 plants edited by CRISPR-Cas9 showed 90%–100% editing efficiency and only 0%–60% efficiency in CRISPR-Cas12a.
As mentioned before, Cas12a nucleases exhibit fluctuating activity depending on temperature, which proves to be a major problem in experimental design. This behavior was demonstrated in AsCas12a, FnCas12a, and LbCas12a in Arabidopsis, rice, and maize. Both AsCas12a and LbCas12a work better at 28°C or above, which is a problem for some model organisms, including plants. An AsCas12a variant called enhanced AsCpf1 (enAsCpf1) demonstrated 2 times higher activity at a lower temperature when active inside human cells. When Schindele and Puchta investigated LbCas12a, enLbCas12a, and temperature tolerant LbCas12a (ttLbCas12a) at 22°C in Arabidopsis, ttLbCas12a exhibited the highest activity of the three.
Overall, the experimental data of various research teams suggests that CRISPR-Cas12a is a strong competitor of CRISPR-Cas9 and could replace it as a more efficient tool for some applications in several model plants.
CRISPR-Cas9 and CRISPR-Cas12a in Animals
Genome editing using CRISPR-Cas systems in animals bears considerable resemblance to the corresponding systems in microorganisms and plants, with a few exceptions.
In the search for a new CRISPR system (other than CRISPR-Cas9) that could be used in Drosophila melanogaster to increase the number of potential target sequences, Port et al. in Heidelberg, Germany, studied multiplexed conditional genome editing employing the CRISPR-Cas12a system. The team demonstrated that LbCas12a, but not AsCas12a, can exhibit robust gene editing in vivo. Since for most crRNAs, LbCas12a activity decreases sharply at low temperatures, gene editing can be controlled by temperature modulation. CRISPR-Cas system taking advantage of LbCas12a can be effectively used for multiplex genome editing in vivo, resulting in loss-of-function phenotypes corresponding to using three crRNAs (per target gene) and achieving similar efficiency to the newest Cas9 system. Gene editing can be performed in a variety of tissues.
One of the experimental results of Port et al. that illustrate the temperature-dependence of Cas12a is shown in Figure 2. The team used two CRISPR-Cas12a systems (plus control) to edit the ebony (e) gene at three different temperatures. The e-loss-of function resulted in Drosophila's dark cuticle. Note that in this experiment, Drosophila edited with LbCas12a exhibited the loss-of-function phenotype for all tested temperatures. On the contrary, Drosophila edited with AsCas12a only exhibited this phenotype for 29°C experimental conditions.
Figure 2. Temperature-dependent editing of ebony
Another field where Cas12a has been gaining increasing popularity compared to Cas9 is base editing. While in plants, no Cas12a-mediated base editing has been demonstrated yet, it has been several years since it was achieved in animal cells. Cas12a is used instead of Cas9 for targeting T-rich regions, as the PAM sequence of Cas9 (G-rich) usually does not allow it to bind to the aforementioned sites. In 2018, the first cytidine deaminase base edit using modified Cas12a was achieved in human cells by Li et al. The team fused a rat APOBEC1 domain and uracil DNA glycosylase inhibitor to dLbCas12a. This base editor was dubbed dLbCpf1-BE0. dLbCpf1-BE0 demonstrated editing effect 8-13 bp before the PAM sequence and demonstrated low levels of undesired indel mutations and non-C-to-T substitutions. The base editing efficiency reached 20-22%. Also, the base editing range (with altered PAM) has been increased by using Acidaminococcus sp. Cas12a variant (enAsCas12a) in human cells.
In 2020, Chen et al. identified another promising Cas12a nuclease ortholog originating from Coprococcus eutactus called CeCas12a and compared its performance to AsCas12a and LbCas12a in human cells. The experimental results suggest that CeCas12a is more stringent when it comes to PAM recognition, both in vitro and in vivo. It also exhibited very low off-target editing rates in cells, especially at C-containing PAMs, while maintaining a comparable efficiency of multiplex editing to AsCas12a and LbCas12a. These features make CeCas12a a promising agent that could satisfy the demanding conditions for approval in therapeutic applications in the future.
Table 1. made by me
Table 2.&3. Adiego-Pérez, B., Randazzo, P., Daran, J. M., Verwaal, R., Roubos, J. A., Daran-Lapujade, P., & van der Oost, J. (2019). Multiplex genome editing of microorganisms using CRISPR-Cas. FEMS microbiology letters, 366(8), fnz086. https://doi.org/10.1093/femsle/fnz086
Table 4.&5. Banakar, R., Schubert, M., Collingwood, M., Vakulskas, C., Eggenberger, A. L., & Wang, K. (2020). Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene. Rice (New York, N.Y.), 13(1), 4. https://doi.org/10.1186/s12284-019-0365-z
Table 6. Alok, A., Sandhya, D., Jogam, P., Rodrigues, V., Bhati, K. K., Sharma, H., & Kumar, J. (2020). The Rise of the CRISPR/Cpf1 System for Efficient Genome Editing in Plants. Frontiers in plant science, 11, 264. https://doi.org/10.3389/fpls.2020.00264
Figure 1: Yao R, Liu D, Jia X, Zheng Y, Liu W, Xiao Y. CRISPR-Cas9/Cas12a biotechnology and application in bacteria. Synth Syst Biotechnol. 2018;3(3):135‐149. Published 2018 Oct 3. doi:10.1016/j.synbio.2018.09.004
Figure 2: Fillip Port, Maja Starostecka, Michael Boutros Multiplexed conditional genome editing with Cas12a in Drosophila (2020) https://doi.org/10.1101/2020.02.26.966333