Lessons in genome engineering: opportunities, tools and pitfalls

Abstract

CRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome. This technology is now routine for many organisms and cell lines. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. We describe how these tools and protocols can be implemented easily and efficiently. Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. Our tools are designed for use in Drosophila but the lessons we draw are likely to be widely relevant. AUTHOR SUMMARYThe genome contains all the information that an organism needs to develop and function throughout its life. One of the goal of genetics is to decipher the role of all the genes (typically several thousands for an animal) present in the genome. One approach is to delete each gene and assay the consequences. Deletion of individual genes is now readily achieved with a technique called CRISPR/Cas9. However, simple genetic deletion provides limited information. Here we describe strains and DNA vectors that streamline the generation of more sophisticated genetic tools. We describe general means of creating alleles (genetic variants) that enable gene activity to be measured and experimentally modulated in space and time. Although the tools we describe are universally applicable, each gene requires special consideration. Based on our experience of successes and failures, we suggest measures to maximise the chances that engineered alleles serve their intended purpose. Although our methods are designed for use in Drosophila, they could be adapted to any organism that is amenable to CRISPR/Cas9 genome modification.

Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition

“…The RPS23[R67K] allele was generated by CRISPR/Cas9-mediated homologous directed repair. Suitable 5’ and 3’ homology arms, as well as the fragment carrying the R67K substitution, were cloned into the pTV attP-Pax-Cherry targeting vector 59 and gRNAs were cloned into the pCFD4 vector (Addgene #49411). The resulting plasmids were co-injected in a nos-Cas9 strain 60 .…”

Rapid and robust optogenetic control of gene expression in Drosophila

“…The Gal4 PAM target (CGG) is located exactly 7bp after aa147, the last aa of the Gal4DBD. To generate pTV-Gal4DBD:2xnMagHigh1, the attP site from the original TV DattP -PAX-Cherry (Poernbacher et al, 2019) was first removed. Using Gibson assembly, Gal4DBD was then inserted (aa1-aa147) fused to 2xnMagHigh1 and followed by the p10 3 0 UTR in the 5 0 MCS.…”

CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

“…If the goal is simply to introduce 24 copies of the MS2 stem-loop sequences, then a one-step CRISPR approach, which directly inserts the MS2 cassette, will be quicker and simpler. Moreover, CRISPR-Cas9 events inserting a sequence into the genome directly, based on a single cut, occur more frequently than the replacement of sequences that include large deletions ( Poernbacher et al., 2019 ). Alternatively, a two-step approach can be used when it is advantageous to repeatedly target the same gene locus.…”

“…(2015) . Experimental designs to insert tags into coding sequences through one-step or two-step CRISPR-Cas9 approaches are summarized by Poernbacher et al. (2019) .…”

“…For the two-step CRISPR approach, try to reduce the amount of sequence deleted, as the efficiency of CRISPR-Cas9 events may depend on the size of the deletion. Even though large deletions of up to 30 kb have been successfully replaced with an attP site, smaller deletions can be obtained with a higher efficiency and limit rearrangements at the edited region ( Poernbacher et al., 2019 ).…”