Homology Arms Research Articles

Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction-modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.

A recombineering system for Bacillus subtilis based on the native phage recombinase pair YqaJ/YqaK

Bacillus subtilis plays an important role in fundamental and applied research, and it has been widely used as a cell factory for the production of enzymes, antimicrobial materials, and chemicals for agriculture, medicine, and industry. However, genetic manipulation tools for B. subtilis have low efficiency. In this work, our goal was to develop a simple recombineering system for B. subtilis. We showed that genome editing can be achieved in B. subtiliis 1A751 through co-expression of YqaJ/YqaK, a native phage recombinase pair found in B. subtilis 168, and the competence master regulator ComK using a double-stranded DNA substrate with short homology arms (100 bp) and a phosphorothioate modification at the 5′-end. Efficient gene knockouts and large DNA insertions were achieved using this new recombineering system in B. subtilis 1A751. As far as we know, this is the first recombineering system using the native phage recombinase pair YqaJ/YqaK in B. subtilis. In conclusion, this new recombineering system provides a simple and fast tool for genetic manipulation of B. subtilis, and it will promote studies of genome function, construction of production strains, and genome mining in B. subtilis.

CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize

Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.

Construction and application of a new CRISPR/Cas12a system in Stenotrophomonas AGS-1 from aerobic granular sludge.

Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Thus, investigating AGS in the aspect of biofilm and microbial attachment at the genetic level would help to reveal the mechanism of granule biofilm formation. In this work, a two-plasmid CRISPR/Cas12a genome editing system was constructed to identify attachment genes for the first time in Stenotrophomonas AGS-1 from AGS. One plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms. Acidaminococcus sp. Cas12a (AsCas12a) was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1. CRISPR/Cas12a-mediated rmlA knockout decreased attachment ability by 38.26%. Overexpression of rmlA in AGS-1 resulted in an increase of 30.33% in attachment ability. These results showed that the modulation of rmlA was an important factor for the biofilm formation of AGS-1. Moreover, two other genes (xanB and rpfF) were knocked out by CRISPR/Cas12a and identified as attachment-related genes in AGS-1. Also, this system could achieve point mutations. These data indicated that the CRISPR/Cas12a system could be an effective molecular platform for attachment gene function identification, which would be useful for the development of AGS in wastewater treatment. This article is protected by copyright. All rights reserved.

Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.

Establishment of CRISPR-Cas9 system in Bifidobacteria animalis AR668

Bifidobacteria are representative intestinal probiotics that have extremely high application value in the food and medical fields. However, the lack of molecular biology tools limits the research on functional genes and mechanisms of bifidobacteria. The application of an accurate and efficient CRISPR system to genome engineering can fill the gap in efficient genetic tools for bifidobacteria. In this study, CRISPR system of B. animalis AR668 was established, which successfully knocked out gene 0348 and gene 0208. The influence of different homology arms and fragments on the knockout effect of the system was explored. In addition, the inducible plasmid curing system of bifidobacteria was innovatively established. This study contributes to the genetic modification and functional mechanism analysis of bifidobacteria.

Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selectedsgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency forco-transfectionof CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editingstrategy could efficiently target the HBB locus and enrich for HDR-positive cells.

Predicting and Improving Insertions by Prime Editing

Predicting and Improving Insertions by Prime Editing

SsDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells

BackgroundRecent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.ResultsIn this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.ConclusionsThese results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors.

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

Immunogenicity and safety of homologous and heterologous booster vaccination of ChAdOx1 nCoV-19 (COVISHIELD™) and BBV152 (COVAXIN®): a non-inferiority phase 4, participant and observer-blinded, randomised study.

Primary SARS-CoV-2 vaccination has been shown to wane with time and provide lower protection from disease with new viral variants, prompting the WHO to recommend the administration of booster doses. We determined the safety and immunogenicity of homologous or heterologous boosters with ChAdOx1 nCoV-19 (COVISHIELD™) or BBV152 (COVAXIN®), the two vaccines used widely for primary immunization in India, in participants who had already received two primary doses of these vaccines. Participants primed with two doses each of COVISHIELD™ or COVAXIN® 12-36 weeks previously, were randomised to receive either COVISHIELD™ or COVAXIN® booster in a 1:1 ratio. The primary outcome was day 28 post-booster anti-spike IgG seropositivity and secondary outcomes were anti-spike IgG levels and assessment of safety and reactogenicity. The results of 90 days intention-to-treat analysis are presented. This trial is registered with ISRCTN (CTRI/2021/08/035648). In the COVISHIELD™ primed group with 200 participants, the seropositivity 28 days post booster in the heterologous COVAXIN® arm was 99% and non-inferior to the homologous COVISHIELD™ arm, which was also 99% (difference 0%; 95% CI:-2.8% to 2.7%). The geometric mean concentration (GMC) of anti-spike antibodies following heterologous COVAXIN® boost on day 28 was 36,190.78 AU/mL (95% CI: 30,526.64-42,905.88) while the GMC following homologous COVISHIELD™ boost was 97,445.09 AU/mL (82,626.97-114,920.7). In the COVAXIN® primed group with 204 participants, the seropositivity 28 days post booster in the heterologous COVISHIELD™ arm was 100% and non inferior to the homologous COVAXIN® arm which was 96% (difference 4%, 95% CI: 0.2%-7.8%). The GMC following heterologous COVISHIELD™ boost was 241,681.6 AU/mL (95% CI: 201,380.2-290,048.3) compared to homologous COVAXIN® boost, which was 48,473.94 AU/mL (95% CI: 38,529.56-60,984.95). The day 28 geometric mean ratio (GMR) of the anti-spike IgG between the heterologous and homologous boosted arms was 0.42 (95% CI: 0.34-0.52) in the COVISHIELD™ primed group and 5.11 (95% CI: 3.83-6.81) in the COVAXIN® primed group. There were no related serious adverse events reported in any group. Homologous and heterologous boosting with COVISHIELD™ or COVAXIN® in COVISHIELD™ or COVAXIN® primed individuals are immunogenic and safe. A heterologous boost with COVISHIELD™ after COVAXIN® prime offers the best immune response among the four combinations evaluated. Azim Premji Foundation and Bill and Melinda Gates Foundation.

Tactics targeting circular mRNA biosynthesis.

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

Long-Read DNA Sequencing: Recent Advances and Remaining Challenges.

DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.

CRISPR-mediated genome editing in poplar issued by efficient transformation.

CRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences. CRISPR-Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone. Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression. Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.

Abstract 1469: Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription

Abstract EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and regulates transcription. Biochemical and depletion studies have demonstrated that EWSR1 regulates basal transcription, and that in Ewing sarcoma (EWS) cells expressing the fusion oncoprotein EWSR1::FLI1, EWSR1 may contribute to the deregulation of gene expression observed in this cancer. To further study EWSR1’s function in Ewing sarcoma, we generated reporter cell lines to visualize and quantify its endogenous expression within EWS cells. To generate EWSR1-fluorescent reporter A673 and TC-32 EWS cell lines, we used CRISPR-Cas9 and sgRNAs targeting the 5’ end of the first exon of EWSR1 and a donor plasmid consisting of the mNeonGreen (mNG) reporter gene, a G4S3 linker, and EWSR1 homology arm templates. Upon validation of successfully modified clones (A673 and TC-32), we used confocal microscopy to confirm EWSR1’s localization to the nucleoplasm. Also, consistent with previous analysis of EWSR1’s protein-protein interactions, we confirmed using immunoprecipitation that the mNG-EWSR1 protein retains its interaction with EWSR1::FLI1 and RNA polymerase II (RNA pol II) - total and phosphorylated (pS5 and pS2). FRAP analysis (Zeiss Airyscan) showed that in cells, EWSR1 exists in two fractions, one that rapidly exchanges (A673 T½: 9.47 secs; TC-32 T½: 6.13 secs) and one that remains bound for a longer duration (A673 T½: 92.15 secs; TC-32; T½: 52.63 secs). We next used structured illumination microscopy to interrogate EWSR1’s spatial organization in EWS cells at a high resolution (~100 nm). First, we determined that EWSR1’s organization within the nucleoplasm is regular as measured by a signal-to-signal distance of ~300 nm in both cell lines (A673 cells 297±6.71 nm; TC-32 cells 304±8.59 nm). Analysis of EWSR1’s spatial organization relative to nucleic acids and proteins that function in gene expression indicates that EWSR1 partially co-localizes with DNA (visualized using DAPI), Histone 3, BRD4 and MED1 (visualized using immunofluorescence - IF). In contrast, we observed EWSR1 colocalizes completely with nascent RNA (visualized using Click-IT RNA Alexa Fluor 488, Invitrogen) (Manders overlap coefficient (MOC) A673: 0.96±0.005; TC-32: 0.97±0.005). Furthermore, we detected over 90% colocalization of concentrated EWSR1 signals (defined by specific fluorescent intensities) with RNA pol II (IF) in states associated with transcriptional initiation (pS5) (MOC A673: 0.94±0.011; TC-32: 0.93±0.02) and elongation (pS2) (MOC A673: 0.95±0.02; TC-32: 0.97±0.06). These studies will aid analysis of changes in EWSR1’s localization within the nucleus and relative to proteins that regulate gene expression following inhibition of transcription, including the application of compounds that disrupt EWSR1::FLI1’s transcriptional activity. Citation Format: Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Patrick Pichling, Katelyn R. Ludwig, Raj Chari, Michael Kruhlak, Jadranka Loncarek, Natasha J. Caplen. Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1469.

Safety and efficacy of COVID-19 prime-boost vaccinations: Homologous BBIBP-CorV versus heterologous BNT162b2 boosters in BBIBP-CorV-primed individuals.

Booster vaccine doses against SARS-CoV-2 have been advocated to address evidence of waning immunity, breakthrough infection, and the emergence of immune-evasive variants. A heterologous prime-boost vaccine strategy may offer advantages over a homologous approach, but the safety and efficacy of this approach with the mRNA vaccine BNT162b2 (BNT: Pfizer) and inactivated BBIBP-CorV (BBIBT: Sinopharm) vaccines have not been studied. We conducted a non-randomized, non-blinded phase II observational community trial acrossBahrain, investigating the reactogenic and immunogenic responseof participants who had previously received two doses of BBIBP, followed by a third booster dose of either BBIBP (homologous booster) or BNT (heterologous booster). Immunogenicity through serological statuswas determined at baseline and on the following 8thweek. Reactogenicity data (safety and adverse events) were collected throughout study period, in addition to participant-led electronic journaling. 305 participants (152 BBIBP and 153 BNT booster) were enrolled in the study,with 246 (127 BBIBP and 119 BNT booster) included in the final analysis. There was a significant increase in anti-SARS-CoV-2 antibody levels post booster administration in both groups; however, the heterologous BNT arm demonstrated a significantly larger mean increase in the level of spike (S) antigen-specific antibodies (32.7-fold increase versus 2.6, p<0.0001) and sVNT neutralising antibodies (3.4-fold increase versus 1.8, p<0.0001), whereas the homologous arm demonstrated a significant increase in the levels of nucleocapsid (N) antigen-specific antibodies (3.8-fold increase versus none). Non-serious adverse events (injection site pain, fever, and fatigue) were more commonly reported in the heterologous arm, but no serious adverse events occurred. Heterologous prime-boost vaccination with the mRNA BNT162b2 (Pfizer) vaccine in those who had received two doses of inactivated virus BBIBP-CorV (Sinopharm) vaccine demonstrated a more robust immune response against SARS-CoV-2 than the homologous BBIBP booster and appears safe and well tolerated. Clinical Trial Registry Number (ClinicalTrials.gov): NCT04993560.

CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae.

Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S.cerevisiae.