Cas9 Nuclease Research Articles

Quadruple adenine base–edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease–engineered T cells

Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.

Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB 3 Variant for Junctional Epidermolysis Bullosa.

Efficient Dual Cas9 Nickase Correction of a Prevalent Pathogenic LAMB 3 Variant for Junctional Epidermolysis Bullosa.

Unravelling the advances of CRISPR-Cas9 as a precise antimicrobial therapy: A systematic review.

Unravelling the advances of CRISPR-Cas9 as a precise antimicrobial therapy: A systematic review.

Agrin-deficient osteocytes disrupt bone tissue homeostasis in male mice.

Agrin-deficient osteocytes disrupt bone tissue homeostasis in male mice.

Dual Cation Exchange coupled with Multi-Modal Chromatography Allows the Production of High-Purity and Low residuals cGMP-Grade Cas9 Nuclease for Cell Therapy Applications

Dual Cation Exchange coupled with Multi-Modal Chromatography Allows the Production of High-Purity and Low residuals cGMP-Grade Cas9 Nuclease for Cell Therapy Applications

Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands

Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands

RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.

To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.

Heritable virus-induced germline editing in tomato.

Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.

Advancements in CRISPR-Cas9 for Fanconi anemia

Fanconi anemia (FA) is a hereditary bone marrow failure syndrome that is characterized by genomic instability and heightened sensitivity to DNA cross-linking agents. In recent years, the CRISPR-Cas9 technology has exhibited groundbreaking progress in the field of gene therapy for FA. The traditional CRISPR-Cas9 technology has been successfully applied in FA gene editing. Further, single-base editing technology, based on the CRISPR/Cas9 system, performs precise and efficient gene repair for prevalent gene mutations in patients with FA. The prime editing technology provides new possibilities for gene editing; however, its application in FA has not been initiated. Despite significant advancements in FA gene editing technology, several challenges remain, including the collection of sufficient hematopoietic stem cells, the risk of increased tumorigenesis postgene editing, chromosomal instability, and off-target effects. Future research is recommended to focus on optimizing sgRNA and Cas9 nucleases, designing stricter PAM sequences to reduce off-target effects, and devising personalized gene editing strategies. Further, ethical and regulatory issues as well as long-term follow-ups are crucial priorities for future gene editing work. With continuous technological advancements and in-depth clinical trials, we expect more breakthroughs in FA treatment using the CRISPR-Cas9 technology in the future. This article reviews the latest research progress of CRISPR technology in FA treatment and analyzes the advantages and disadvantages of this technology in FA gene therapy.

An accelerated transgene-free genome editing system using microparticle bombardment of sorghum immature embryos

Abstract The key factors for genome-editing in plants using CRISPR/Cas9, such as the Cas9 nuclease and guide RNA (gRNA) are typically expressed from a construct that is integrated into the plant genome. However, the presence of foreign DNA in the host genome causes genetic and regulatory concerns, particularly for commercialization. To address this issue, we developed an accelerated pipeline for generating transgene-free genome-edited sorghum (Sorghum bicolor) in the T0 generation. For proof-of-concept, we selected the Phytoene desaturase (PDS) gene as the target due to its visible phenotype (albinism) upon mutation. Following microprojectile-mediated co-transformation with a maize (Zea mays)-optimized Cas9 vector and a guide RNA (gRNA) cassette with a geneticin (G418) resistance gene, we divided tissue derived from immature embryos into two groups (with and without antibiotic selection) and cultured them separately as parallel experiments. In regenerated plants cultured on medium containing MS basal nutrition (to allow albino plants to survive), we detected higher rates of albinism in the non-selection group, achieving editing rates of 11.1–14.3% compared with 4.2–8.3% in the antibiotic selection group. In the T0 generation, 22.2–38.1% of albino plants from the non-selection group were identified as transgene-free, whereas only 0–5.9% from the selection group were transgene-free. Therefore, our strategy efficiently produced transgene-free genome-edited plants without the need for self-crossing or outcrossing, demonstrating the feasibility of achieving transgene-free genome-edited sorghum plants within a single generation. These findings pave the way for commercializing transgene-free genome-edited lines, particularly for vegetatively propagated crops like pineapple, sugarcane, and banana.

Engineering of SauriCas9 with enhanced specificity.

SauriCas9 is a compact Cas9 nuclease showing promise for invivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor Tcell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.

Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model.

Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model.

High-accuracy crRNA array assembly strategy for multiplex CRISPR.

High-accuracy crRNA array assembly strategy for multiplex CRISPR.

Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing.

CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5'-NGG-3' for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5'-NAA-3' and 5'-NGAA-3' PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans.

The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.

The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases. Our data suggest site-specific DNA cleavage mostly occurs in a stepwise fashion. Indels dominate the induced mutations. A few, unexpected on-site large deletions (>1 kb) are also observed. Notably, genome-wide analyses reveal no off-target mutations. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progeny. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes involved in human neurodegenerative diseases.

Simplified Protocol for the Purification of Native Cas Nucleases for DNA-Free Genome Editing.

DNA-free genome editing by the direct delivery of CRISPR-associated nucleases has emerged as a promising technology due to its precision and reduced risk of off-target effects. However, existing purification protocols for native Cas proteins require the use of complex instrumentation, which limits their application. Here, we present a simplified protocol for the purification of native Cas9, Cas12RR and dCas9-VP64 nucleases optimized for DNA-free genome editing. Our approach leverages a streamlined affinity and ion exchange chromatography coupled with minimal downstream processing, ensuring a good yield and activity of the purified proteins. The in vitro analysis of the purified ribonucleoprotein complex demonstrated a good efficiency of DNA target cleavage. This simplified protocol increases the opportunity to adopt CRISPR technology, and enables broader access to DNA-free genome editing tools also for laboratories that are not specifically equipped for protein purification.

Nano-Polymers as Cas9 Inhibitors.

Despite wide applications of CRISPR/Cas9 technology, effective approaches for CRISPR delivery with functional control are limited. In an attempt to develop a nanoscale CRSIPR/Cas9 delivery platform, we discovered that several biocompatible polymers, including polymalic acid (PMLA), polyglutamic acid (PGA), and polyaspartic acid (PLD), when conjugated with a trileucine (LLL) moiety, can effectively inhibit Cas9 nuclease function. The Cas9 inhibition by those polymers is dose-dependent, with varying efficiency to achieve 100% inhibition. Further biophysical studies revealed that PMLA-LLL directly binds the Cas9 protein, resulting in a substantial decrease in Cas9/sgRNA binding affinity. Transmission electron microscopy and molecular docking were performed to provide a possible binding mechanism for PMLA-LLL to interact with Cas9. This work identified a new class of Cas9 inhibitor in nano-polymer form. These biodegradable polymers may serve as novel Cas9 delivery vehicles with a potential to enhance the precision of Cas9-mediated gene editing.

Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.

Cleavable donor-assisted CRISPR/Cas9 system significantly improves the efficiency of large DNA insertion in Physcomitrium patens.

Precise insertion of desired fragments can be achieved by CRISPR/Cas9-based genome editing. However, a decrease in knock-in efficiency has been observed with increasing length of exogenous inserts. In this study, we developed an in vivo cleavable (IVC) donor-assisted CRISPR/Cas9 system to improve efficiency, particularly for larger inserts, in the moss Physcomitrium patens (P. patens). The IVC donor, which contains two Cas9 nuclease recognition sites flanking the homology template, enables the in vivo release of the linear template for homology-directed repair (HDR) when co-delivered with the corresponding CRISPR/Cas9 plasmid into protoplasts. In our experimental framework, two distinct sgRNAs and four different DNA inserts were evaluated. Compared with standard circular donors, IVC donors significantly enhanced the efficiency of CRISPR/Cas9-mediated precise insertion of 5.8, 7.5, and 11.1 kb DNA fragments at the PpPDV2-4 sgRNA target site, improving integration rates from 29.6 to 67.8%, from 15.0 to 72.0%, and from 12.1 to 65.6%, respectively. At an alternative sgRNA2 target site within the Pp6c18_3160 locus, the IVC donor also demonstrated a higher knock-in efficiency for a 7.4 kb fragment compared with the standard circular donor. This IVC donor-assisted CRISPR/Cas9 approach for large fragment knock-in represents a powerful tool for basic research and synthetic biology efforts in moss species. Moreover, this strategy may be potentially applicable to crops that are amenable to protoplast transformation and regeneration, facilitating the improvement of key traits.

In vitro gene editing using primary cells derived from Cas9-expressing pigs.

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has significantly facilitated the generation of gene-edited (GE) pigs. Although GE pigs are promising for agricultural and biomedical applications, the entire process of generating useful GE pigs is time- and labor-intensive. To overcome this, in vivo gene-editing techniques have been developed, where Cas9 nuclease and single guide RNA (sgRNA) are directly injected into animals; however, their efficiency remains low owing to the large size of the nuclease. In this study, we generated a Cas9-expressing pig by inserting the Cas9 gene into the ROSA26 locus, resulting in its constitutive expression in various tissues. We also confirmed the pig's fertility. In vitro experiments with primary cells from the pig confirmed effective gene deletion by adding only sgRNAs. These results suggest that the Cas9-expressing pig generated in this study could serve as an effective platform for in vivo and in vitro gene editing in agricultural and biomedical research.