SiT-Cas12a Multiplex Genome Editing
Last update: Oct 2020
This section provides an overview of the study conducted by the scientists at ETH Zurich and the University of Groningen who managed to fit up to 25 crRNAs and a Cas12a nuclease transcript on a single mRNA and employ it in human genome editing. They named their system SiT-Cas12a (single-transcript Cas12a) and demonstrated that it can be employed for gene knockout as well as transcription activation/repression for up to 15 genes simultaneously.
The work of Carlo C. Campa, Niels R. Weisbach António J. Santinha, Danny Incarnato and Randall J. Platt titled "Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts" was published in 2019 in Nature Methods. It can be accessed on Research Gate (offsite link).
Encoding of Cas12a and crRNAs on a Single Transcript
The works preceding this publication showed that the CRISPR-Cas12a system could be optimized by the shortening of direct repeats (DRs), which are part of the CRISPR array and are used for the binding between crRNA and Cas12a nuclease. Another study employed different promoters that orchestrate the transcription of Cas12a and pre-crRNA. The paper by Campa et al. improves upon those strategies in order to enable efficient multiplex genome editing in mammalian cells.
All the following experiments were performed on the human embryonic kidney (HEK) 293T cells.
In the first experiment, the researchers confirmed that both Cas12a and CRISPR arrays, which consist of spacers (used for targeting DNA sequences) and direct repeats (DRs), can both be encoded on a single Pol II- derived transcript. Verifying this feature was necessary because, in the case of mammals, different promoters drive transcription of different RNA molecules. Pol II promoters usually orchestrate transcription of coding genes that produce long RNA sequences, while Pol III promoters are used for the production of small non-coding RNAs, including crRNAs. If we want to place both Cas12a and CRISPR arrays in a single construct (in a single plasmid), we need to make sure that the entire construct can be expressed using the same promoter.
There was also another challenge that needed to be addressed by the researchers. Overall, the construct is made up of a promoter region followed by a Cas12a transcript followed by a CRISPR array region followed by a polyadenylation (poly(A)) tail that stabilizes the entire structure. When the pre-crRNA inside the CRISPR array is cut and processed into mature crRNA, poly(A) tail is removed. This decreases the stability of the remaining Cas12a transcript by exposing it to the activity of 3′–5′ exonucleases. Thus, the scientists had to find a way to prevent the Cas12a portion of the transcript from getting destabilized by the pre-crRNA processing.
According to the experimental results, the stability of a protein (such as Cas12a) encoded upstream of the CRISPR array can be increased by placing a tertiary structural RNA motif consisting of a triple helix called Triplex in between the protein and CRISPR array codes. Triplex is a 110-nucleotide long structure, originally obtained from the 3′ end of the mouse non-coding RNA Metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Triplex has been demonstrated to stabilize mRNA transcripts lacking poly(A) tails. Thus, even when the crRNA-making and poly(A) portions of the transcript are removed, the Cas12a part of the transcript, which is still attached to the Triplex, can remain stable and proceed to be translated into a functional nuclease. Overall, Triplex contributes to improved pre-crRNA processing and Cas12a production.
The structure of the transcript that showed the best performance in this experiment is presented in Figure 1. EF1a, at the beginning of the transcript, is an RNA polymerase Pol II promoter. Cas12a transcript is displayed as the yellow box. Directly downstream of Cas12a transcript is the purple Triplex stabilizing the mRNA of this protein. Next, we have direct repeats in grey and a spacer sequence in blue, together making up the pre-crRNA (CRISPR array) portion of the transcript. In the later part of the experiment, Campa et al. attach more DRs and spacers to this region, which makes multiplex genome editing possible. After DR, we can see the poly(A) tail, which stabilizes the entire transcript until the pre-crRNA region is processed.
Thus, the researchers developed the SiTCas12a, which is short for single-transcript Cas12a, encoded on a single Pol II-driven mRNA. It has the structure outlined above, and it uses Acidaminococcus sp. Cas12a (AsCas12a) nuclease variant. The researchers confirmed that the platform could perform efficient (single-gene) genome editing, including conditional and inducible edits.
Figure.1 Encoding of Cas12a and CRISPR array on a single transcript
SiT-Cas12a in Multiplex Genome Editing
The next step was to test SiT-Cas12a in multiplex genome editing. First, the team investigated the genome editing efficiency of knocking out 5 genes: FANCF1, EMX1, GRIN2B, VEGF, and DNMT1. Next, they placed the spacers targeting all 5 genes into a CRISPR array inside of the SiTCas12a transcript. The structure of the transcript is shown in Fig.2.a (top). Multiplex genome editing efficiencies for the 5 genes are displayed in Fig.2.b. The y-axis is % of indels, which corresponds to the number of successful knockouts. On top of evaluating the performance of SiT-Cas12a, researchers have also tested a regular Cas12a-based transcript without the Triplex (control). RNase dead Cas12a, which cannot process its pre-crRNA, and DNase dead Cas12a, which cannot make double strand breaks (DSBs), were the extra controls. As expected, their genome editing efficiencies were close to zero. Fig.2.b shows that the % of indels was the highest for SiT-Cas12a, ranging between 5% and 23%.
Figure 2. Multiplex genome editing of 5 genes using SiT-Cas12a
Campa et al. also tested SiT-Cas12a integrated platform against experimental designs relying on the independent transcription of Cas12a and a CRISPR array from distinct promoters. Editing efficiencies were higher or comparable to other platforms, suggesting that SiT-Cas12a could be a viable alternative to other Cas12a platforms in the context of multiplex genome editing.
After that, the researchers decided to test the editing efficiency of gene knockout when several distinct crRNAs target different regions within that very same gene. They placed 10 spacers inside the CRISPR array of SiT-Cas12a platform, each spacer targeting a distinct region of CD47 locus. As a result, gene editing efficiency reached 60%. In comparison, while crRNAs were used one by one, the editing efficiency was 2 to 17%. These experimental results show that by employing several crRNAs targeting different parts of the same gene, we can obtain better knockout efficiencies compared to using one crRNA per gene. This technique was then used to create triple knockouts, which shows it can be used in multiplex editing.
Multiplexed Transcriptional Regulation Using SiT-Cas12a
It turns out that the Sit-Cas12a platform can be used not only for genome editing but also for transcription regulation. The CRISPR array part of the transcript does not change, but the Cas12a transcript is changed to that of ddCas12a (DNase dead). In addition, ddCas12a is fused to a transcription effector. In the case of gene repression, one to three copies of Krüppel associated box (KRAB) domain (transcription repressor) were fused to the C-terminus of ddCas12a. Attaching three KRAB domains has been demonstrated to yield the highest gene repression levels compared to other combinations. Using this experimental design, the team introduced 20 spacers targeting 10 different genes (2 crRNAs per gene) into the SiT-Cas12a platform. As you can see in Fig.3 featuring relative gene expression on the y-axis, the original expression level being equal to 1, multiplex gene repression reduced gene expression levels to between 5% to 70% of the original expression values
Figure 3. Multiplex gene repression of 10 genes using SiT-Cas12a
Multiplexed gene activation is achieved using a very similar experimental design, but instead of KRAB domains, ddCas12a is fused, for example, to VPR activator or p65 activation domain (p65) + heat shock factor 1 (HSF1). Out of several combinations tested, 3 sp65p + HSF1 activator domain yielded the highest gene activation levels, denoted as relative gene expression. Similarly to repression experiments, the SiT-ddCas12a platform was equipped with a CRISPR array consisting of 20 spacers targeting 10 genes. As shown in Fig.4, all genes exhibited around 10 to 1000-fold increase in expression.
These experimental results show the potential of SiT-ddCas12a in transcriptional regulation.
Figure 4. Multiplex gene activation of 10 genes using SiT-Cas12a
Orthogonal Gene Editing and Transcriptional Control Using SiTCas12a
One interesting feature that the last set of experiments took advantage of is that a shortened gRNA (crRNA) results in a nuclease binding to, but not cleaving the target sequence. This is true both for Cas9 and Cas12a, and it is the result of unique DNA binding kinetics. It means that even when we are using a single SiT-Cas12a platform with functional (not ddCas12a) nuclease and an effector domain, we could perhaps control which genes to edit, and which genes to repress/activate simply by placing spacers of different length inside of the CRISPR array.
For the following experiments, the research team also used AsCas12a. The CRISPR arrays contained both long (20 bp) and short (15 bp) spacers. The mechanism underlying these orthogonal transcriptional regulation and gene editing experiments is presented in Fig.5. As before, the SiT-Cas12a platform contains a Pol II promoter arrow followed by Cas12a transcript. The ED box in grey stands for the effector domain and encodes either transcription repressor or activator domain(s). In purple, we can see the stabilizing Triplex, followed by CRISPR array consisting of direct repeats in grey and 15 bp long and 20 bp long spacer sequences. We can see that the SiT-Cas12a platform gives rise to a catalytically active Cas12a nuclease bound to the effector domain, and shorter and longer mature crRNAs. crRNAs associate with Cas12a, forming a Cas12a-[ED]–crRNA complex. It goes on to search the cell's DNA for the sequence complementary to the crRNAs. The complexes with shorter crRNAs cannot make DSBs, but stay bound to DNA, and the effector domains fused to Cas12a can control the gene transcription. At the same time, the complexes containing longer crRNAs can make DSBs and thus perform genome editing successfully.
The team had to then assess how well this model works in an experimental setting. First, they used DNase active and inactive (dead) Cas12a nucleases fused to transcription repressors or activators (ED). These SiT-Cas12a-[ED] platforms also contained CRISPR arrays consisting either of short or long spacers, which were targeting 2 different promoters. They tested editing and transcription control of RAB5A and PIK3C3 genes in editing/repression, and ASCL1 and IL1B genes in editing/activation experiments. As expected, only DNase active SiT-Cas12a-[ED] combined with longer spacers resulted in gene editing (knockout), while the platforms employing ddCas12a were unsuccessful at making indels. The key experimental result is that the SiT-Cas12a-[ED] combined with shorter spacers prompted gene repression or gene activation. No gene editing was detected. These observations suggest that even though a DNase-active Cas12a is normally able to make DSBs, when it is bound to a shorter instead of a longer crRNA, it will not cleave the DNA. The repression and activation activity observed in the experiments was the result of Cas12a being fused to the aforementioned effector domains. Overall, these experimental results support the model outlined in Fig.5 and demonstrate that we can control whether a gene will be edited or repressed/activated simply by changing the spacer length.
Figure 5. Structure of the SiT-Cas12a platform for gene regulation/editing
Finally, the team had to demonstrate that this is also possible for multiplex gene manipulation, and not just for single genes like in the experiment outlined before. They designed a large CRISPR array, which contained both short spacers for targeting 10 genes whose transcription was to be regulated, and long spacers for targeting 5 genes for knockout. Overall, the CRISPR array consisted of 25 spacers and was expressed together with the SiT-Cas12a-[ED] transcript. Fig.6 shows the experimental results. On the left (Fig.6.a), we see the results for the multiplex gene expression and knockout (SiT-Cas12a-[Repr]), and on the right (Fig.6.b), we see the results for the multiplex gene activation and knockout (SiT-Cas12a-[Activ]). Top y-axes are relative gene expression; bottom y-axes are % indels. As you can see on the left, SiT-Cas12a-[Repr] platform was successful at reducing gene expression of 10 genes by around 50% on average and made indels in all 5 genes, albeit with low efficiency. Looking on the right, we can observe that SiT-Cas12a-[Activ] platform increased gene expression on average 10-fold for 9 genes and as much as 1000 fold for IL1B. The gene editing efficiency of this platform was slightly better than that of the system on the left, with around 15 % indels generated for the 5 genes tested.
Figure 6. Multiplexed orthogonal gene regulation/editing
In this study, the team has created the SiT-Cas12a platform, in which a single Pol II promoter expresses a single transcript that contains both Cas12a and CRISPR array. This platform simplifies the experimental design, as up to now, Cas nucleases and gRNAs were expressed using different promoters. SiT-Cas12a platform was shown to host up to 25 crRNAs, which allows for targeting of up to 25 DNA sequences. The team speculates that in the future, it could be possible to target hundreds of sequences using this method.
As many genes may contribute to a single condition or phenotype, being able to modulate them all simultaneously is crucial if we want to grasp those conditions fully. It is thus all the more important to find ways of placing dozens of gRNAs on a single plasmid, in order to simplify the experimental designs. The SiT-Cas12a platform described by Campa et al. could bring us one step closer to exploring the full potential of Cas12a nuclease in this context.
AsCas12a nuclease used in this study is known to have high-temperature requirements (around 37°C), which is appropriate for editing genomes of mammalian cells, such as human cells used in this study. However, since plants are usually reared at around 25°C, this experimental design could be difficult to reproduce in plant genome engineering. One possible solution to that would be using a Cas12a protein with lower temperature requirements, such as LbCas12a. This might make it possible to set up a SiT-Cas12a platform in plants.
 Campa, C.C., Weisbach, N.R., Santinha, A.J. et al. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods 16, 887–893 (2019). https://doi.org/10.1038/s41592-019-0508-6
 Wilusz, J. E., Jn Baptiste, C. K., Lu, L. Y., Kuhn, C. D., Joshua-Tor, L., & Sharp, P. A. (2012). A triple helix stabilizes the 3' ends of long non-coding RNAs that lack poly(A) tails. Genes & development, 26(21), 2392–2407. https://doi.org/10.1101/gad.204438.112
Campa, C.C., Weisbach, N.R., Santinha, A.J. et al. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods 16, 887–893 (2019). https://doi.org/10.1038/s41592-019-0508-6