CRISPR-Cas9 human gene replacement and phenomic characterization inCaenorhabditis elegansto understand the functional conservation of human genes and decipher variants of uncertain significance

Abstract

Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole exome and whole genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision.We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene Phosphatase and Tensin Homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosenory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E) and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss-of-function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance. remarkably efficient and robust in C. elegans (Dickinson and Goldstein, 2016; Norris et 70 al., 2015). 71Here, we present a broadly applicable strategy that adapts CRISPR-Cas9 genome 72 engineering for targeted replacement of C. elegans genes with human genes. We illustrate 73 how the library of knockout and humanized transgenics generated with this approach can 74 be efficiently combined with automated machine vision phenotyping to rapidly discover 75 novel gene functions, assess the functional conservation of human genes, and how this 76 will allow for analysis of the effects of variants of uncertain significance with 77 unprecedented precision. It is our hope that the human gene replacement and phenomic 78 characterization strategy delineated in this article will serve both basic and health 79 researchers alike, by serving as an open and shareable resource that will aid any genome 80 engineer interested in understanding the functional conservation of human genes, and the 81 functional consequences of their variants. 82 83 Results 84A general genome editing strategy for direct replacement of a C. elegans gene with a 85 single copy of its human ortholog 86To replace the Open Reading Frame (ORF) of an orthologous gene with a human 87 gene ...

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

“…However, the challenge currently lies in inserting larger fragments of DNA, such as the integration of fluorescent reporters into endogenous loci or gene replacements. Even though it has been demonstrated that this can be achieved by using targeting vectors with long homology arms (Dickinson et al 2013;Schwartz and Jorgensen 2016;McDiarmid et al 2018), plasmid construction usually involves cumbersome cloning steps. An alternative option is to use a PCR product with 35-bp flanking sites that are homologous to the insertion site as a repair template (Paix et al 2015).…”

Systematic phenomics analysis of ASD-associated genes reveals shared functions and parallel networks underlying reversible impairments in habituation learning

“…Our data adds to the recent and rapidly expanding use of model organism phenomics to discover the functions of poorly characterized genes identified through genomic sequencing. 44,101,[153][154][155][156][157][158] We have shown how such data can be used to identify novel genetic interactions, establish variant functional assays, and develop tests of phenotypic reversibility.…”

“…49,141 We have also recently shown that directly replacing daf-18 with only a single copy of its human ASD-associated ortholog PTEN at the endogenous locus using CRISPR-Cas9 is able to rescue multiple sensory abnormalities caused by complete deletion of daf-18. 142 A recent systematic humanization effort of 414 genes in yeast showed that >50% of human genes could functionally replace their yeast ortholog. 143 Indeed, at this point it would not be surprising if a highly conserved human gene could replace its C. elegans ortholog.…”

“…[36][37][38][39][40][41][42][43] There are clear C. elegans orthologs for >50% of human genes, and human genes have repeatedly been shown to be so structurally and functionally conserved that they can directly replace their C. elegans ortholog. 36,[44][45][46][47][48][49] There are well-annotated libraries of C. elegans strains with deletion alleles available for >16,000 of the ~20,000 protein coding genes. [50][51][52] C. elegans small size and rapid hermaphroditic mode of reproduction (3 days from egg to egg-laying adult) allows for the routine cultivation of large numbers of isogenic animals.…”

“…Further, CRISPR-Cas9 genome engineering has proven to be reliable and efficient in C. elegans, and unlike most organisms analyzed to date, multiple rigorous whole-genome sequencing studies have revealed no evidence of significant off-target effects due to CRISPR-Cas9 genome editing in this organism. 44,[53][54][55][56][57][58][59] Finally, we developed the Multi-Worm Tracker (MWT), a machine vision system that allows for comprehensive phenotypic analysis of large populations of freely behaving animals while they perform complex sensory and learning behaviors. 60,61 Multiplexing by running several trackers in parallel allows for analysis of multiple measures of morphology, locomotion, mechanosensory sensitivity, as well as several forms of learning in hundreds of animals simultaneously.…”