A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity

Abstract

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

Biosynthesis of Circular RNA ciRS-7/CDR1as Is Mediated by Mammalian-wide Interspersed Repeats

“…The gRNAs (Figure 2C) are listed in Table S1. The annealed gRNAs were subcloned into the BpiI-cleaved (Thermo Fisher Scientific) site of a modified version of CRSPR vector px458 (#43138, Addgene) without the ITR element that expresses Cas9/EGFP (Højland Knudsen et al, 2018). To increase the efficiency and accuracy of the CRISPR targeting site (MIR elements), single-stranded donor oligodeoxyribonucleotides (ssODN) acting as repair templates were used (Table S1).…”

Optimized Transgene Delivery Using Third‐Generation Lentiviruses

“…Due to their broad tropism, lentiviruses can be used to deliver genetic material into a variety of cell types with high efficiency, including post-mitotic neurons (Naldini et al, 1996;Zufferey et al, 1997). Because of their long-term and stable integration capability, lentiviruses can be used to generate stable cell lines with an antibiotic selection cassette (LaGory et al, 2015) or reporter (Knudsen et al, 2018), providing an indispensable tool to elucidate gene function in development and disease. Lentiviruses can be packaged with shorthairpin RNA (for knock-down experiments), the open reading frame of a gene of interest, and the CRISPR-Cas9 system with multiple guide RNAs (Yiu, Tieu, Nguyen, Wong, & Smit-McBride, 2016) for genome editing.…”