Developmental Biology

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1^fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress Ca_V2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic Ca_V2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease.

Key features

• Virus-mediated genetic perturbation in neonatal and young adult mice.

• Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

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.

Graphical overview

Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR–based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement.

Key features

• This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish.

• Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening.

• Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT.

Graphical overview

Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com)

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish.

Graphic Abstract:

GeneWeld method for CRISPR/Cas9 targeted integration.