The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.

Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.

In the originally published version of this article, the DNA sequence reported in the Experimental Procedures for the PQR in Drosophila cells was the incorrect variant. The correct DNA sequence for the PQR in Drosophila cells is 5'-GGAAGCGGAGAAGGTCGTGGTAGT CTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT-3'.

Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein-expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein. Expression levels can be predetermined by selecting promoters from genes with the desired level of expression. To quantify recombinant protein expression, a protein quantitation reporter (PQR) is incorporated between the endogenous and foreign genes. The PQR allows equimolar production of the endogenous protein, the recombinant protein, and a fluorescent reporter. As a result, expression levels of both the endogenous and recombinant proteins can be continuously monitored using fluorescence.