Novel Cas9 Effectors With Robust Function In Human Cells

Introduction: The characterization of CRISPR-Cas systems and the development of Cas-based genome editing technologies which function in human cells has revolutionized biotechnology, offering considerable potential for therapeutic genome and epigenome editing. Despite the abundance of CRISPR-Cas systems in bacterial species, only a limited number are effective in human cells. This provides limited opportunities to use alternative systems to circumvent challenges in the translational pathway or provide orthogonal regulation. To expand the CRISPR-Cas toolbox, we predicted and tested 47 Cas9 effector proteins and their cognate single-guide RNAs (sgRNAs) from diverse type II CRISPR-Cas loci. Among these, four showed robust and highly specific gene repression in human cells when used as nuclease null dCas9s fused with a KRAB domain. All four Cas9s also exhibited efficient nuclease activity in human cells. We further examined gene activation when fused with p300, and observed efficient gene activation with two dCas9s. Notably, our most active Cas9 exhibited comparable performance to the benchmark S. aureus and S. pyogenes Cas9s widely used for repression, activation, and nuclease activity. This comprehensive exploration expands the repertoire of CRISPR-Cas9 systems, addressing critical challenges in advancing effective genome editing for therapeutic applications.

Materials and

Methods: Cas9 Identification and Prediction
All available prokaryotic genomes were downloaded from the National Center for Biotechnology Information’s Reference Sequence Database. CRISPR arrays were identified using the CRISPR Visualizer software package. Cas genes were annotated using the CRISPRdisco software pipeline. To determine spacer identity, all spacer sequences were compared against NCBI’s Nucleotide Collection database, and PAMs were determined by inspection of the alignments.

Repression of HBE in K562 Cells
K562 HBE-mCherry cells (40000) were transduced with dCas9-KRAB lentivirus (50µL) corresponding to each dCas9 in 500µL RPMI with 5µg/mL polybrene. Three days later, the resulting dCas9-KRAB cell line was further transduced with sgRNA lentivirus. Then 9 or 10 days post-transduction, cells were harvested and analyzed for mCherry repression on a flow Sony SH800 cytometer.

Transfection of HEK293T cells
Plasmids encoding Cas9(or dCas9-p300) and sgRNA were transfected into HEK293T cells using lipofectamine 3000. After 72 hours, gDNA or RNA was extracted from transfected cells. Extracted gDNA was subjected to PCR to generate amplicons flanking each target site and sequenced on an Illumina MiSeq. Extracted RNA was subjected to RT-qPCR for HBG expression.

PCSK9 repression in AML12 cells
AML-12 cells were transduced with lentivirus encoding dCas9-KRAB-2A-GFP-2A-BlastR. GFP-expressing cells were isolated via cell sorting. To assay PSCK9 repression in each cell line, cells were transduced with sgRNA virus and harvested after 9 days. RNA was extracted from frozen cell pellets and subjected to RT-qPCR for PCSK9.

Results, Conclusions, and Discussions: In order to expand the CRISPR-Cas toolkit, we used the CRISPRdisco pipeline to mine non-pathogenic and non-commensal bacterial genomes for CRISPR-Cas9 systems. We then adapted these systems for mammalian use by predicting PAM sequences and crRNA:tracrRNA sequences to generate sgRNAs from dual guide RNAs. The Cas9 proteins were converted into inactivated versions (dCas9) and genetically fused with the KRAB repressor domain to perform epigenetic gene repression experiments in human cells. To assess efficacy in repressing an endogenous gene, we used a K562 cell line in which the HBE gene is tagged with an in-frame mCherry reporter protein, facilitating rapid evaluation of HBE knockdown. Based on robust repression in our HBE repression assay, we selected four Cas9 systems for further characterization.

To evaluate nuclease activity in human cells, plasmids encoding the wild-type, nuclease-active Cas9 proteins and sgRNA targeting HBE were transfected into HEK293T cells. Deep sequencing of targets sites demonstrated that all four tested Cas9s are effective nucleases in mammalian cells. Further testing of the most efficient Cas9 targeting the TRAC CDS showed editing rates of 35%. To demonstrate additional epigenome editing activities of these newly characterized dCas9 proteins, we generated fusions to the catalytic core of histone acetyltransferase p300 and assessed activation of the HBG1 gene in 293T cells and observed gene activation with two of four Cas9s. We selected PCSK9 as an example therapeutically relevant target for gene repression. Repression of PCSK9 in the liver has been shown to reduce cholesterol levels and is a promising target for treatment of hereditary hypercholesterolemia. The most efficient novel dCas9-KRAB repressed PCSK9 in AML12 cells to levels comparable to those achieved with S. aureus dCas9-KRAB. In conclusion, we have demonstrated that 4 previously uncharacterized Cas9 systems are novel and useful additions to the CRISPR toolkit.

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