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Ittiprasert, Wannaporn, Chawalit Chatupheeraphat, Victoria H. Mann, Wenhui Li, André Miller, Taiwo Ogunbayo, Kenny Tran, Yousef N. Alrefaei, Margaret Mentink-Kane, and Paul J. Brindley. "RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni." International Journal of Molecular Sciences 23, no. 2 (January 6, 2022): 631. http://dx.doi.org/10.3390/ijms23020631.
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The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.
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Shebanova, Regina, Natalia Nikitchina, Nikita Shebanov, Vladimir Mekler, Konstantin Kuznedelov, Egor Ulashchik, Ruslan Vasilev, et al. "Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA." Nucleic Acids Research 50, no. 2 (December 24, 2021): 1162–73. http://dx.doi.org/10.1093/nar/gkab1227.
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Abstract CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.
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Petiwala, Sakina, Apexa Modi, Tifani Anton, Erin Murphy, Sabah Kadri, Hengcheng Hu, Charles Lu, Michael J. Flister, and Daniel Verduzco. "Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays." CRISPR Journal 6, no. 1 (February 1, 2023): 75–82. http://dx.doi.org/10.1089/crispr.2022.0093.
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Sousa, Patricia, Tusneem Janoudi, Edouard deDreuzy, Mark S. Shearman, Kate Zhang, and Kai-Hsin Chang. "Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia." Blood 138, Supplement 1 (November 5, 2021): 1858. http://dx.doi.org/10.1182/blood-2021-149956.
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Abstract Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy. EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny. To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production. In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. Disclosures Sousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.
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Pattali, Rithu, Kaitlyn Izzo, Edward Goncz, Steven Sexton, Kevin Wasko, John Zuris, Michael Nehil, et al. "191 GAPDH knock-in of high affinity CD16 in iPSC derived NK cells drives high-level expression and increased anti-tumor function." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A203. http://dx.doi.org/10.1136/jitc-2021-sitc2021.191.
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BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.
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Lee Yu, Henson, Yumeng Cao, Xiao Lu, and I.-Ming Hsing. "Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity." Biosensors and Bioelectronics 189 (October 2021): 113382. http://dx.doi.org/10.1016/j.bios.2021.113382.
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Marino, Nicole D., Jenny Y. Zhang, Adair L. Borges, Alexander A. Sousa, Lina M. Leon, Benjamin J. Rauch, Russell T. Walton, et al. "Discovery of widespread type I and type V CRISPR-Cas inhibitors." Science 362, no. 6411 (September 6, 2018): 240–42. http://dx.doi.org/10.1126/science.aau5174.
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Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here, we report the discovery of 12 acr genes, including inhibitors of type V-A and I-C CRISPR systems. AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs—including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a—when assayed in human cells. The acr genes reported here provide useful biotechnological tools and mark the discovery of acr loci in many bacteria and phages.
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Yang, Zhao, Kun Zhang, Tianying Xing, Suhang Bai, Zongyi Shen, Luyao Wang, Lingzhi Wang, Zichen Zhang, Chong Li, and Wei Zhang. "Application of Clustered Regularly Interspaced Short Palindromic Repeat—Cas12a System in Cancer Research and its Structural Basis." Cancer Plus 4, no. 1 (January 25, 2022): 30. http://dx.doi.org/10.18063/cp.v4i1.240.
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The clustered regularly interspaced short palindromic repeat-Cas12a (CRISPR-Cas12a) system is a new type of CRISPR-Cas system. As a unitary effector protein in this system, Cas12a recognizes 5’-TTTN-3’ protospacer-adjacent motif and exhibits cleavage activity of double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), supplementing the toolbox of CRISPR system. Compared to CRISPR-Cas9 system, CRISPR-Cas12a system has the advantage of high specificity, which is a promising tool for genetic manipulation in the basic cancer research and clinical cancer therapy. To date, three Cas12a proteins including Acidaminococcus sp. Cas12a (AsCas12a), Francisella novicida Cas12a (FnCas12a), and Lachnospiraceae bacterium Cas12a (LpCas12a) have been applied in transcriptional regulation or genome editing through CRISPR RNAs complementary to target DNA or RNA in cancer cells or immune cells. This review summarizes the latest applications of CRISPR-Cas12a system in cancer research and its structural basis.
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Vasilev, Ruslan, Natalia Gunitseva, Regina Shebanova, Aleksei Korzhenkov, Anna Vlaskina, Marta Evteeva, Irina Polushkina, et al. "Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii." International Journal of Molecular Sciences 23, no. 16 (August 18, 2022): 9289. http://dx.doi.org/10.3390/ijms23169289.
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Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5′-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.
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Ma, Shufeng, Xinlong Wang, Yongfei Hu, Jie Lv, Chengfang Liu, Kaitong Liao, Xiaohua Guo, Dong Wang, Ying Lin, and Zhili Rong. "Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain." Nucleic Acids Research 48, no. 18 (September 28, 2020): 10590–601. http://dx.doi.org/10.1093/nar/gkaa779.
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Abstract The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.
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Murugan, Karthik, Arun S. Seetharam, Andrew J. Severin, and Dipali G. Sashital. "CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects." Journal of Biological Chemistry 295, no. 17 (March 11, 2020): 5538–53. http://dx.doi.org/10.1074/jbc.ra120.012933.
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Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.
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Zhang, Liyang, H. Tomas Rube, Christopher A. Vakulskas, Mark A. Behlke, Harmen J. Bussemaker, and Miles A. Pufall. "Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes." Nucleic Acids Research 48, no. 9 (April 21, 2020): 5037–53. http://dx.doi.org/10.1093/nar/gkaa231.
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Abstract CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.
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Wettengel, Jochen M., Lea Hansen-Palmus, Sofiya Yusova, Lauren Rust, Sreya Biswas, Julien Carson, Junghyun Ryu, Benjamin N. Bimber, Jon D. Hennebold, and Benjamin J. Burwitz. "A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing." International Journal of Molecular Sciences 24, no. 9 (May 5, 2023): 8271. http://dx.doi.org/10.3390/ijms24098271.
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CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
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Allen, Alexander G., Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, et al. "Abstract 562: AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model." Cancer Research 82, no. 12_Supplement (June 15, 2022): 562. http://dx.doi.org/10.1158/1538-7445.am2022-562.
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Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. CAR-T cell therapies, however, have limited efficacy in solid tumors and are associated with severe toxicity, highlighting the need for safer and more efficacious novel cell therapies. With their intrinsic tumor killing capacity, few treatment-related toxicities, and the ability to be given to patients off-the shelf, natural killer (NK) cells are an attractive alternative therapy option to CAR-T cells. While most NK cell therapies are produced from healthy donor cells, deriving NK cells from induced pluripotent stem cells (iPSCs) has the unique advantage that a clone with any desired edits can be generated. We aim to leverage our iPSC platform in combination with our proprietary gene editing technologies to create highly differentiated off-the-shelf treatments for solid tumors. Using our proprietary engineered AsCas12a, we generated double knocked-in (DKI) iPSC clones in which a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into the GAPDH locus to increase the effector function and persistence of iNKs. Constitutive surface expression of CD16 and mbIL-15 by the DKI iNKs was demonstrated. DKI iNKs showed significantly increased natural and antibody dependent cellular cytotoxicity when compared to wild type (WT) iNKs in a SKOV3 tumor spheroid assay in vitro. Furthermore, in the absence of exogeneous cytokines, DKI iNKs persistence in vitro was dramatically improved over WT iNKs. The anti-tumor efficacy of the DKI iNKs in vivo was evaluated using a SKOV3 ovarian cancer model. Tumor bearing mice were treated with WT or DKI iNKs intraperitoneally in combination with trastuzumab or treated with trastuzumab alone. No exogenous cytokines were administered. DKI iNKs combined with a single dose or multiple doses of trastuzumab exerted greater tumor control compared to WT iNKs with trastuzumab, or trastuzumab alone. A single dose of DKI iNKs combined with three doses of trastuzumab induced tumor clearance in 6 out of 8 mice and significantly prolonged survival. Importantly, DKI iNKs were detected in the peritoneum of the treated animals for greater than 3 months, demonstrating that the mbIL-15 maintained iNK survival for a prolonged period of time in the absence of exogeneous cytokine support. In summary, knocking-in CD16 and mbIL-15 to the GAPDH locus of iPSCs dramatically increased the persistence of the DKI iNKs which exhibited robust anti-tumor activities in a solid tumor mouse model. These data demonstrate that our platform enables the development of off-the-shelf iNK cell medicines that may be highly effective for treating solid tumors. Citation Format: Alexander G. Allen, Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, Stephen Sherman, Laura Blaha, Mark Shearman, Kate Zhang, Kai-Hsin Chang. AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 562.
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Zhu, Dan, Junyi Wang, Di Yang, Jianzhong Xi, and Juan Li. "High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity." International Journal of Molecular Sciences 22, no. 24 (December 10, 2021): 13301. http://dx.doi.org/10.3390/ijms222413301.
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CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position −3 for PAM TATV and at position −4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.
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Hanna, Rabi, Haydar Frangoul, Christopher Mckinney, Luis Pineiro, Markus Mapara, Kai-Hsin Chang, Michael Jaskolka, et al. "S264: EDIT-301 SHOWS PROMISING PRELIMINARY SAFETY AND EFFICACY RESULTS IN THE PHASE I/II CLINICAL TRIAL (RUBY) OF PATIENTS WITH SEVERE SICKLE CELL DISEASE USING HIGHLY SPECIFIC AND EFFICIENT ASCAS12A ENZYME." HemaSphere 7 (August 2023): e05170e0. http://dx.doi.org/10.1097/01.hs9.0000967968.05170.e0.
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Gerew, Alexandra, Steven Sexton, Kevin M. Wasko, Mark S. Shearman, Kate Zhang, Kai-Hsin Chang, and Samia Q. Khan. "Deletion of CISH and TGFβR2 in iPSC-Derived NK Cells Promotes High Cytotoxicity and Enhances In Vivo Tumor Killing." Blood 138, Supplement 1 (November 5, 2021): 2780. http://dx.doi.org/10.1182/blood-2021-150731.
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Abstract Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells. Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity. We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing. In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. Disclosures Gerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.
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Maerken, Melanie, David MacEwan, Nicholas Harper, Joseph R. Slupsky, and Adam Linley. "Gene Editing of BTK in Acute Myeloid Leukaemia Using CRISPR-Cas9." Blood 132, Supplement 1 (November 29, 2018): 3952. http://dx.doi.org/10.1182/blood-2018-99-113804.
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Abstract Acute myeloid leukemia (AML) blood cancer is found primarily in adults and the elderly, being the third biggest blood cancer killer. In the last decades there has been some improved rates in younger patients, but little improvement in older patients. AML survival rates are the worst of all blood cancers, therefore finding new therapeutic targets to treat AML is crucial. Bruton's tyrosine kinase (BTK) is involved in the signalling of multiple receptors including growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors and integrins. BTK in turn activates many of the major downstream signalling pathways that control cell migration, adhesion, survival and proliferation. BTK is best known for its role in B-cell receptor signalling, but BTK is also expressed and constitutively active in AML cell lines and AML patient blasts. Targeting BTK in B-cell malignancies with the BTK inhibitor ibrutinib has shown clinical effectiveness and tolerability in recent patient trials. Ibrutinib is orally active and covalently binds at the Cys-481 in the ATP-binding domain of BTK, to irreversibly block its enzymatic activity. A proportion of CLL and MCL patients treated with ibrutinib relapse during the therapy, with secondary resistance developing against ibrutinib. This resistance is mainly due to a BTK cysteine to serine mutation at position 481 (C481S) which disrupts ibrutinib's covalent binding to BTK. As little is known about BTK's role and therapeutic potential in AML, we aimed to study the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib. We furthermore generated the BTK-C481S mutation in AML cell lines using the CRISPR-Cas9 gene-editing system. Shown here for the first time is a gene-edited mutation introduced into a human leukemia cell clone that has been purified to homogeneity. We explored the functional impact of BTK-C481S mutation on ibrutinib sensitivity, and mutation-induced adaptation of the phosphoproteome and kinome in AML cells. HEK-293T cells were used for validation purposes to find the most efficient knock-in strategy. We tested the most commonly used CRISPR endonuclease spCas9 as well as two other Cas9 orthologs called SaCas9 and AsCas12a. The designed spacer sequences within the sgRNA that target the gene of interest were delivered either via plasmid or in vitro transcripts. A repair template for homology directed repair (HDR) was designed containing the necessary base changes to change the amino acid at position 481 in BTK from cysteine to serine. The HDR template was delivered via transfection as single-stranded oligodeoxynucleotide (ssODN) or incorporated into plasmid. All strategies tested were successful but due to the poor transfectability of AML cell lines, we used lentiviral delivery to introduce BTK-C481S mutation into AML cell lines. The BTK-C481S mutation was introduced in THP-1 and OCI-AML3 cells. Mutant-positive cells were found via clonal selection and Sanger sequencing. Wild-type (WT) as well as mutant cells were treated with various ibrutinib concentrations ranging from 1 nM to 1 µM and analysed for BTK expression/phosphorylation via Western blot, with effects on cell proliferation, viability and cell cycle measured via flow cytometry. We also performed phosphoproteomics and kinome assays comparing untreated as well as treated (30 nM ibrutinib, 2h) THP-1 WT and THP-1 BTK-C481S cells. Western blot results showed that the effective concentration required for BTK inhibition is 30-fold higher in BTK-C481S mutant compared to WT cells (300 nM vs 10 nM). Wash-out experiments revealed that ibrutinib binding can be reversed in BTK-C481S mutant cells but not WT cells. Little effect of ibrutinib on proliferation and viability were seen comparing BTK-C481S mutant and WT AML cells. These signalling adaptations in BTK-C481S cells are consistent with changes observed in ibrutinib-resistant leukemia patient samples. Disclosures Slupsky: Verastem: Research Funding.
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Allen, Alexander G., Rithu Pattali, Kaitlyn M. Izzo, Jared A. Getgano, Kevin M. Wasko, Laura C. Blaha, John A. Zuris, Kate Zhang, Mark S. Shearman, and Kai-Hsin Chang. "A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence." Blood 138, Supplement 1 (November 5, 2021): 4809. http://dx.doi.org/10.1182/blood-2021-153258.
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Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.
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Cetin, Ronay, Martin Wegner, Leah Luwisch, Sarada Saud, Tatjana Achmedov, Sebastian Süsser, Antonella Vera-Guapi, et al. "Optimized metrics for orthogonal combinatorial CRISPR screens." Scientific Reports 13, no. 1 (May 6, 2023). http://dx.doi.org/10.1038/s41598-023-34597-8.
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AbstractCRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.
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Jacobsen, Thomas, Chunyu Liao, and Chase L. Beisel. "The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs." FEMS Microbiology Letters 366, no. 8 (April 1, 2019). http://dx.doi.org/10.1093/femsle/fnz085.
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ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.
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Zhang, Liyang, John A. Zuris, Ramya Viswanathan, Jasmine N. Edelstein, Rolf Turk, Bernice Thommandru, H. Tomas Rube, et al. "AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines." Nature Communications 12, no. 1 (June 23, 2021). http://dx.doi.org/10.1038/s41467-021-24017-8.
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AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.
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Zhang, Yingxiao, Yanhao Cheng, Hong Fang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, Randall P. Niedz, James N. Culver, and Yiping Qi. "Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases." Frontiers in Genome Editing 4 (January 31, 2022). http://dx.doi.org/10.3389/fgeed.2022.780238.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.
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An, Yi, Ya Geng, Junguang Yao, Chunxiang Fu, Mengzhu Lu, Chun Wang, and Juan Du. "Efficient Genome Editing in Populus Using CRISPR/Cas12a." Frontiers in Plant Science 11 (November 19, 2020). http://dx.doi.org/10.3389/fpls.2020.593938.
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The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.
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Zhu, Zhijian, Manyu Zhang, Dandan Liu, Defei Liu, Tao Sun, Yujing Yang, Jiacheng Dong, et al. "Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants." Microbial Cell Factories 22, no. 1 (August 11, 2023). http://dx.doi.org/10.1186/s12934-023-02149-4.
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Abstract Background Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. Results In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. Conclusions Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.
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Krishnamurthy, Sateesh, Christine Wohlford-Lenane, Suhas Kandimalla, Gilles Sartre, David K. Meyerholz, Vanessa Théberge, Stéphanie Hallée, et al. "Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia." Nature Communications 10, no. 1 (October 28, 2019). http://dx.doi.org/10.1038/s41467-019-12922-y.
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Abstract The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.
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Maule, Giulia, Antonio Casini, Claudia Montagna, Anabela S. Ramalho, Kris De Boeck, Zeger Debyser, Marianne S. Carlon, Gianluca Petris, and Anna Cereseto. "Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing." Nature Communications 10, no. 1 (August 7, 2019). http://dx.doi.org/10.1038/s41467-019-11454-9.
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Abstract Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272–26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.
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Illa-Berenguer, Eudald, Peter R. LaFayette, and Wayne A. Parrott. "Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice." Frontiers in Genome Editing 5 (March 17, 2023). http://dx.doi.org/10.3389/fgeed.2023.1074641.
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The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.
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DeWeirdt, Peter C., Kendall R. Sanson, Annabel K. Sangree, Mudra Hegde, Ruth E. Hanna, Marissa N. Feeley, Audrey L. Griffith, et al. "Optimization of AsCas12a for combinatorial genetic screens in human cells." Nature Biotechnology, July 13, 2020. http://dx.doi.org/10.1038/s41587-020-0600-6.
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Okada, Satoshi, Goro Doi, Shitomi Nakagawa, Emiko Kusumoto, and Takashi Ito. "Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae." G3 Genes|Genomes|Genetics, August 30, 2021. http://dx.doi.org/10.1093/g3journal/jkab304.
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Abstract Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.
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Zhang, Liyang, John A. Zuris, Ramya Viswanathan, Jasmine N. Edelstein, Rolf Turk, Bernice Thommandru, H. Tomas Rube, et al. "Author Correction: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines." Nature Communications 12, no. 1 (July 19, 2021). http://dx.doi.org/10.1038/s41467-021-24770-w.
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Maule, Giulia, Antonio Casini, Claudia Montagna, Anabela S. Ramalho, Kris De Boeck, Zeger Debyser, Marianne S. Carlon, Gianluca Petris, and Anna Cereseto. "Author Correction: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing." Nature Communications 11, no. 1 (October 22, 2020). http://dx.doi.org/10.1038/s41467-020-19351-2.
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Knott, Gavin J., Brady F. Cress, Jun-Jie Liu, Brittney W. Thornton, Rachel J. Lew, Basem Al-Shayeb, Daniel J. Rosenberg, et al. "Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a." eLife 8 (August 9, 2019). http://dx.doi.org/10.7554/elife.49110.
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CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.
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Ageely, Eman A., Ramadevi Chilamkurthy, Sunit Jana, Leonora Abdullahu, Daniel O’Reilly, Philip J. Jensik, Masad J. Damha, and Keith T. Gagnon. "Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles." Nature Communications 12, no. 1 (November 15, 2021). http://dx.doi.org/10.1038/s41467-021-26989-z.
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AbstractCRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.
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Wang, Yao, Tao Qi, Jingtong Liu, Yuan Yang, Ziwen Wang, Ying Wang, Tianyi Wang, et al. "A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing." Science Advances 9, no. 6 (February 10, 2023). http://dx.doi.org/10.1126/sciadv.abo6405.
Full textAbstract:
The CRISPR-Cas system can treat autosomal dominant diseases by nonhomologous end joining (NHEJ) gene disruption of mutant alleles. However, many single-nucleotide mutations cannot be discriminated from wild-type alleles by current CRISPR-Cas systems. Here, we functionally screened six Cas12j nucleases and determined Cas12j-8 as an ideal genome editor with a hypercompact size. Cas12j-8 displayed comparable activity to AsCas12a and Un1Cas12f1. Cas12j-8 is a highly specific nuclease sensitive to single-nucleotide mismatches in the protospacer adjacent motif (PAM)–proximal region. We experimentally proved that Cas12j-8 enabled allele-specific disruption of genes with a single-nucleotide polymorphism (SNP). Cas12j-8 recognizes a simple TTN PAM that provides for high target site density. In silico analysis reveals that Cas12j-8 enables allele-specific disruption of 25,931 clinically relevant variants in the ClinVar database, and 485,130,147 SNPs in the dbSNP database. Therefore, Cas12j-8 would be particularly suitable for therapeutic applications.
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Ooi, Kean Hean, Mengying Mandy Liu, Jie Wen Douglas Tay, Seok Yee Teo, Pornchai Kaewsapsak, Shengyang Jin, Chun Kiat Lee, et al. "An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing." Nature Communications 12, no. 1 (March 19, 2021). http://dx.doi.org/10.1038/s41467-021-21996-6.
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AbstractExtensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.
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Kim, Do Yon, Jeong Mi Lee, Su Bin Moon, Hyun Jung Chin, Seyeon Park, Youjung Lim, Daesik Kim, Taeyoung Koo, Jeong-Heon Ko, and Yong-Sam Kim. "Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus." Nature Biotechnology, September 2, 2021. http://dx.doi.org/10.1038/s41587-021-01009-z.
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AbstractGene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.
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Sun, Ruirui, Yuqian Zhao, Wenjuan Wang, Jun-Jie Gogo Liu, and Chunlai Chen. "Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage." Chemical Science, 2023. http://dx.doi.org/10.1039/d2sc05463a.
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Banakar, Raviraj, Mollie Schubert, Gavin Kurgan, Krishan Mohan Rai, Sarah F. Beaudoin, Michael A. Collingwood, Christopher A. Vakulskas, Kan Wang, and Feng Zhang. "Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants." Frontiers in Genome Editing 3 (January 12, 2022). http://dx.doi.org/10.3389/fgeed.2021.760820.
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Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.
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Wang, Yanchun, Shuli Sang, Xin Zhang, Haoxia Tao, Qing Guan, and Chunjie Liu. "Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis." Frontiers in Bioengineering and Biotechnology 9 (January 14, 2022). http://dx.doi.org/10.3389/fbioe.2021.825493.
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A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.
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Bigelyte, Greta, Joshua K. Young, Tautvydas Karvelis, Karolina Budre, Rimante Zedaveinyte, Vesna Djukanovic, Elizabeth Van Ginkel, et al. "Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells." Nature Communications 12, no. 1 (October 26, 2021). http://dx.doi.org/10.1038/s41467-021-26469-4.
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AbstractClass 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.
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Huang, Hongxin, Weiqi Lv, Jinhe Li, Guanjie Huang, Zhihong Tan, Yongfei Hu, Shufeng Ma, Xin Zhang, Linxuan Huang, and Ying Lin. "Comparison of DNA targeting CRISPR editors in human cells." Cell & Bioscience 13, no. 1 (January 16, 2023). http://dx.doi.org/10.1186/s13578-023-00958-z.
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Abstract Background Profiling and comparing the performance of current widely used DNA targeting CRISPR systems provide the basic information for the gene-editing toolkit and can be a useful resource for this field. In the current study, we made a parallel comparison between the recently reported miniature Cas12f1 (Un1Cas12f1 and AsCas12f1) and the widely used Cas12a and Cas9 nucleases in mammalian cells. Results We found that as a CRISPRa activator, Un1Cas12f1 could induce gene expression with a comparable level to that of Cas12a and Cas9, while as a DNA cleavage editor, Cas12f1 exhibited similar properties to Cas12a, like high specificity and dominantly induced deletions over insertions, but with less activity. In contrast, wild-type SpCas9 showed the highest activity, lowest specificity, and induced balanced deletions and insertions. Thus, Cas12f1 is recommended for gene-activation-based applications, Cas12a is for therapy applications, and wild-type Cas9 is for in vitro and animal investigations. Conclusion The comparison provided the editing properties of the widely used DNA-targeting CRISPR systems in the gene-editing field.