Author: İrem Sinem Acınan,
Editor: Ece Güven,
Redactor: Ece Odabaşı
Generating synthetic transcription factors for targeting different loci became much easier via clustered regularly interspersed palindromic repeats (CRISPR) systems. As known, CRISPR systems provided a variety of enzymes like CRISPR associated protein 12a (Cas12a), an RNA-guided homing endonuclease. Cas12a had some advantageous features over Cas9, which is more frequently utilised endonuclease in researches, such as being targeted by single CRISPR RNA (crRNA) instead of requiring a combined crRNA and tracrRNA (trans-activating crRNA), unlike Cas9. Also, Cas12a has RNase activity as well as the ability to recognize and process an array of crRNAs within a single transcript, which provides targeting of the protein to multiple unique loci.
First of all, targeting more synthetic transcription factors to specific genomic regions is necessary for the multiplexed targeting. Especially, for gene network manipulations where multiple promoters are targeted, optimal transactivation is seen for a narrow 350 nucleotide (nt) window within the promoter region. In this study, Bryson et al., reported that targeting more than one copy of the engineered DNase inactive Cas12a (dCas12a) based synthetic transcription factor to the same promoter can enable enhanced transactivation in mammalian cells.
For adaptation, different Cas12a systems were screened with three Cas12a variants from Acidaminococcus sp. (As), Francisella novicida (Fn) and Lachnospiraceae bacterium (Lb), which were fused to the VPR domain of an activator. Lb and As variants were chosen due to their well characterization while the ability of Fn variant to target a wide range of protospacer adjacent motif (PAM) sequence made it a good candidate. After dual luciferase assay of the variants alongside dCas9-VPR, the targeted Fn variant (dFnCas12a-VPR) represented significant transactivation.
By doing orthogonality (cross-reactivity) test for relative transactivation of combinations of each variants with the three targeting crRNAs, researchers concluded that dFnCas12a-VPR and dLbCas12a-VPR represent a good orthogonal pair. Furthermore, the researchers tried to transactivate three endogenous genes, HBB, ASCL1 and IL1RN, as they have previously shown to be notably available for transactivation when targeted with dCas9 based transcription factors. For that, six crRNAs were designed to target three promoters and to utilise a “TTV” PAM sequence within a window 50–300 nt upstream of the transcription start site (TSS). Then, single crRNA plasmids were transfected into HEK293 cells along with dFnCas12a-VPR. Analysis of gene expression by qRT-PCR showed that a single crRNA was enough to enable transactivation of the endogenous genes. Nevertheless, targeting multiple crRNAs to the same promoter enhanced gene expression. The researchers further tested the ability of dFnCas12a-VPR to utilise and process pairs of crRNAs as a single array in which two targeting crRNAs are merged in tandem. They found that a single array consisting of paired crRNAs induced significant upregulation and the pairs synergistically increased the gene expression.
Coding sequence of dFnCas12a-VPR is too long to package into Adeno-associated viruses (AAV). Therefore, a chimeric split-intein (a protein excised for protein splicing) system was utilised, which contains the N-terminal fragment of Npu (Nostoc punctiforme PCC73102) and the C-terminal fragment of SspC (Synechocystis sp. PCC6803) (NpuN-SspC). This system should have expressed the dFnCas12a-VPR coding sequence separately as halves from two constructs before trans-splicing in vivo. The accomplished test showed successful reconstitution of the split-intein dFnCas12a-VPR after trans-splicing for multiplexed transactivation of human genes.
In conclusion, this study suggested that dFnCas12a fused with the VPR domain can be used as a synthetic transcription factor in mammalian cells for easy and simultaneous multiplexed activation of endogenous genes. Also, split-intein versions of genetic constructs can reduce the size of coding sequences, allowing them to be packaged into therapeutically relevant but highly size constrained AAV vectors. Finally, easy and cheap construction of compact crRNA arrays from short oligonucleotides can broaden CRISPR-based applications.
Reference: Bryson, J. W., Auxillos, J. Y., & Rosser, S. J. (2021). Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor. Nucleic Acids Research. https://doi.org/10.1093/nar/gkab1191
– Scientific News Series of Bioinfocodes-
News Articles have been written by our team members through the review and assessment of scientific researches published in 20> Impact Factor Q1 journals (click for the list).