Molecular Biology

This protocol describes the generation of chimeric mice in which the Y chromosome is deleted from a proportion of blood cells. This model recapitulates the phenomenon of hematopoietic mosaic loss of Y chromosome (mLOY), which is frequently observed in the blood of aged men. To construct mice with hematopoietic Y chromosome loss, lineage-negative cells are isolated from the bone marrow of ROSA26-Cas9 knock-in mice. These cells are transduced with a lentivirus vector encoding a guide RNA (gRNA) that targets multiple repeats of the Y chromosome centromere, effectively removing the Y chromosome. These cells are then transplanted into lethally irradiated wildtype C57BL6 mice. Control gRNAs are designed to target either no specific region or the fourth intron of Actin gene. Transduced cells are tracked by measuring the fraction of blood cells expressing the virally encoded reporter gene tRFP. This model represents a clinically relevant model of hematopoietic mosaic loss of Y chromosome, which can be used to study the impact of mLOY on various age-related diseases.

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The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38–68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9^D10A nickase with a pair of PAM-out sgRNAs at a distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy.

Graphical overview

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-N^intein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.

CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.

The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing signals in the vector and endogenous splicing signals in a cloned genomic DNA fragment that contains one or more introns and exons. Minigene-based assay has been used extensively to test the effect of mutations in the splicing of a target sequence. They can also be used to test the ability of CRISPR/Cas9 and one or more associated gRNAs to target specific sequences in the minigene, and determine the effect of these editing events on splicing. As an example, it is shown that CRISPR/Cas9-based, targeted excision of short intronic sequences containing mutations which create cryptic splice signals, can restore normal splicing in a CFTR Hybrid minigene.

Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are now revolutionizing genome editing. The CRISPR/Cas9 system from Streptococcus pyogenes that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. In this protocol, we described the general procedure for CRISPR/Cas9-mediated ssDNA recombineering in C. glutamicum. Small modifications can be introduced into the C. glutamicum chromosome with a high editing efficiency up to 90%.

Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days.

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.

The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more directed approaches. Gene function–and the correlation between phenotype and genotype is most obviously studied with genetics, and it is therefore critical to develop techniques permitting rapid and facile strain construction. Many new and candidate archaeal lineages have recently been discovered, but experimental, genetic access to archaeal genomes is currently limited to a few model organisms. The results obtained from manipulating the genomes of these genetically-accessible organisms have already had profound effects on our understanding of archaeal physiology and information processing systems, and these continued studies also help resolve phylogenetic reconstruction of the tree of life. The hyperthermophilic, planktonic, marine heterotrophic archaeon Thermococcus kodakarensis, has emerged as an ideal genetic system with a suite of techniques available to add or delete encoded activities, or modify expression of genes in vivo. We outline here techniques to rapidly and markerlessly delete a single, or repetitively delete several, continuous sequences from the T. kodakarensis genome. Our procedure includes details on the construction of the plasmid DNA necessary for transformation that directs, via homologous recombination, integration into the genome, identification of strains that have incorporated plasmid sequences (termed intermediate strains), and confirmation of plasmid excision, leading to deletion of the target gene in final strains. Near identical procedures can be employed to modify, rather than delete, a genomic locus.

Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We describe the use (dubbed Multiplexing of Cas9 at Artificial Loci) of installed artificial Cas9 target sequences into the yeast genome that allow for (i) multiplexing with a single sgRNA; (ii) a reduction/elimination in possible off-target effects, and (iii) precise control of the placement of the intended target sequence(s).