Orthogonal transcriptional modulation and gene editing using multiple CRISPR/Cas systems

CRISPR/Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein (RNP) delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated sgRNAs to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein:sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing kn..., Plasmid cloning 
All plasmids were designed based on a backbone suitable for in vitro mRNA transcription previously described in Jensen et al. (2021).22 This plasmid contains a T7 promotor followed by a 45bp 5’UTR ending with a Kozak sequence after which the GOI is inserted. The GOI is followed by a 93bp 3′ UTR of the murine Hba-a1 gene and a 50nt poly A tail and a unique restriction site for run-off IVT. The GOIs for CRISPRa experiments were as follows: dSpCas9-VPR from Jensen et al. (2021), dSpCas9 for dSpCas9-VPRmini and dSpCas9-p65p3-HSF1 was amplified from the dSpCas9-VPR plasmid.22 VPRmini was amplified from Addgene plasmid 99698. pAAV-SCP1-dSa VPR mini.-2X snRP-1 BsaI gRNA was a gift from George Church (Addgene plasmid # 99698 ; http://n2t.net/addgene:99698 ; RRID:Addgene_99698). sp65p3-HSF1 was amplified from Addgene plasmid 129136. pCE059-SiT-Cas12a-[Activ] was a gift from Randall Platt (Addgene plasmid # 128136 ; http://n2t.net/addgene:128136 ; RRID:Addgene_128136). dSpCas9-S..., , # Data files from the \"Orthogonal transcriptional modulation and gene editing using multiple CRISPR/Cas systems\" article

[https://doi.org/10.5061/dryad.r4xgxd2q0](https://doi.org/10.5061/dryad.r4xgxd2q0)

## Description of the data and file structure

**RNA-seq data:**

*Naming of RNA-seq samples:*

CD123_CD5: Jurkat cells treated with simultaneous CRISPRa (dSpCas9-VPR mRNA + CD123 sgRNAs) and CRISPRi (dSaCas9-KRAB mRNA CD5 sgRNAs)

VPR_KRAB: Jurkat cells treated with dSpCas9-VPR and dSaCas9-KRAB mRNA

Sp_Sa: Jurkat cells treated with dSpCas9 and dSaCas9 mRNA

The samples are performed in triplicates (hence the numbers 1, 2 and 3 after sample name)

**RNA-seq analysis**

Jurkat cells were electroporated in biological triplicates for each condition as described previously. The RNA amounts used for each condition in the electroporation mix were: (1) 0.095 µg/µL dSpCas9 + 0.095 µg/µL dSaCas9; (2) 0.095 µg/µL dSpCas9-VPR + 0.095 µg/µL dSaCas9-KOX1; and (3) 0.095 µg/µL dSpCas9-VPR + 0.095 µ...