A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

Abstract

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

High Density Domain-Focused CRISPR Screens Reveal Novel Epigenetic Regulators ofHOX/MEISActivation in Acute Myeloid Leukemia

“…We then wanted to assess the effect of SGF29 deletion in a human AML patient cells. For this, we used AML393 cells, derived from an AML patient; these cells express split-Cas9 and can be used to perform gene-editing in a human AML sample (35). In AML393 cells, SGF29 knockout using the BFP-co-expressing sgRNAs led to a striking reduction in proliferative advantage in a competition assay compared to non SGF29-sgRNA expressing cells (Fig.…”

Genome editing

“…In difficult to transfect cells such as human iPSCs, a piggyBac transposon system provided sustained expression of Cas9 prime editors, a critical factor when combined with optimal pegRNA template design, for efficient gene editing 29 . Recovery of patient derived CRISPR-Cas9 knockout leukemia cells was significantly improved by the development of a fluorescent reporter system that provides a readout of cells harboring high levels of gene editing 38 .…”

“…These powerful Cas9-based genome editing tools allow targeted modification of nearly any gene of interest across most biological systems, enabling molecular genetic studies in organisms in which gene manipulation and transgenesis has traditionally been difficult 25,26 . Key considerations in the application of this technology are optimal delivery and expression of CRISPR/Cas9 reagents.This Collection gathers 17 contributions describing new methods to improve CRISPR/Cas9 genome editing efficiency in a variety of animal and plant systems [27][28][29][30][31][32][33][34] , and its application to develop new genetic tools and models [35][36][37][38] and to gain novel insight into mechanisms regulating organismal physiology, cell behavior, and gene expression [39][40][41][42][43] .The relatively low efficiency of CRISPR/Cas9 targeted integration of exogenous DNA via HDR has been a limiting factor in precision knock-in in mammalian cells and in vivo systems in which the predominant DNA repair pathway is Non-Homologous End Joining (NHEJ). Optimization of each parameter in CRISPR knock-in experimental design, including the specific Cas endonuclease, single or double-stranded DNA template, linear or circular templates, and homology arm length increases the chances of recovering precision HDR edits.…”

“…This Collection gathers 17 contributions describing new methods to improve CRISPR/Cas9 genome editing efficiency in a variety of animal and plant systems [27][28][29][30][31][32][33][34] , and its application to develop new genetic tools and models [35][36][37][38] and to gain novel insight into mechanisms regulating organismal physiology, cell behavior, and gene expression [39][40][41][42][43] .…”

“…www.nature.com/scientificreports/ derived CRISPR-Cas9 knockout leukemia cells was significantly improved by the development of a fluorescent reporter system that provides a readout of cells harboring high levels of gene editing 38 . The Cas9 targetable genome has been significantly expanded with the development of near-PAM-less Cas9 variants that recognize NG, NA, NT and NC PAMs.…”