RNA ; CRISPR-Cas Systems

Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.
CRISPR-Cas Systems ; Bacteria

Animals ; CRISPR-Cas Systems ; Gene Editing ; Humans

CRISPR/Cas(the clustered regularly interspaced short palindromic repeats-CRISPR associated)system exists in most bacteria and all archaea. It is an important strategy for bacteria and archaea to resist foreign nucleic acid invasion and use for self-defense. The CRISPR/Cas system is a simple, fast, and specific diagnostic tool, which is widely used in agriculture, industry, animal husbandry, and medicine. This article mainly introduces and discusses recently advantages and limitations of biosensors combining CRISPR/Cas system with fluorescence, visualization and surface enhanced raman related technologies, as well as future research directions.
Animals ; CRISPR-Cas Systems ; Bacteria/genetics* ; Archaea

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
Humans ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics*

Clustered regulatory interspaced short palindromic repeats (CRISPR) found in bacteria and archaea genome that contains multiple short repeats loci, provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. The first inkling of this hot new genetic engineering tool turned up in 1987, when a research team observed an oddly repetitive sequence at one end of a bacterial gene. Now three types of CRISPR/Cas system have been identified: types I, II and III. In the type II CRISPR/Cas9 system, short segments of foreign DNA termed 'spacers' are integrated within the CRISPR genomic loci, transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and direct sequence-specific cleavage in that a double-strand break (DSB) is generated by Cas proteins. Based on these findings, various genetic methods, including gene targeting (Gene disruption), gene insertion, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This review summarizes recent progress, discusses the prospects of CRISPR/Cas9 system, with an emphasis on its structure, principle, applications and potential challenges, and provides a useful reference for researchers who are interested in this new technique.
Bacteria ; CRISPR-Cas Systems ; DNA ; Genetic Engineering ; Genomics ; RNA

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; Homologous Recombination

Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.
Animals ; Bacteria ; CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Humans ; Mice