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

Abstract

Summary CRISPR-Cas9 genome editing has transformed biology by enabling site-specific genome modifications to be simply engineered. Here, we describe two CRISPR-Cas9 approaches to introduce MS2 stem-loop sequences into endogenous gene loci in Drosophila. This can facilitate live imaging of nascent transcription in Drosophila . For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020) .

Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila

“…The sog attP CRISPR Drosophila line was generated by CRISPR/Cas9 with homology directed repair (HDR) in a two-step CRISPR approach (Baena-Lopez et al, 2013; Hoppe & Ashe, 2021). For a detailed outline of the strategy see (Hoppe & Ashe, 2021). PAM sites (NGG) located either side of the sog start codon were identified using the CRISPR OptimalTarget Finder tool on the flyCRISPR website (Gratz et al, 2014).…”

“…For oligonucleotides sequences encoding sense and antisense strands for guide sequence see Table S1. Complementary guide oligonucleotides were annealed and inserted into the pU6-Bbs1-gRNA plasmid (RRID:Addgene_45946, (Gratz et al, 2013)) as described (Hoppe & Ashe, 2021). Homology arm (HA) sequences were obtained from Drosophila genomic DNA (BL51324) by PCR.…”

Expanding Gene-Editing Potential in Crop Improvement with Pangenomes

“…The most common window sizes for base editors ranges from 4-5 bp to 50-150 bp, allowing for punctual modifications in the target [149][150][151]. Nonetheless, larger cassette insertions have been reported in plants through the use of different CRISPR methods such as non-homologous end joining (NHEJ) that have allowed insertions and deletions that are a few kilobases in size [152][153][154]. However, efficiency drops steeply as the size of the edit increases [155].…”

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

“…This is especially important when testing, for example, mutant transgenes in follow up experiments. Note: Detailed CRISPR protocols have been described ( Hoppe and Ashe, 2021a ; Yu et al., 2021 ), including methods for scarless editing ( Lamb et al., 2017 ; Nyberg et al., 2020 ). …”

“…Note: Detailed CRISPR protocols have been described ( Hoppe and Ashe, 2021a ; Yu et al., 2021 ), including methods for scarless editing ( Lamb et al., 2017 ; Nyberg et al., 2020 ).…”