Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models.

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

Efficient targeted insertion of large DNA fragments without DNA donors.

Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.

Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics.

African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle-antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrument-dependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.