Efficient Multi‐Allelic Genome Editing of Primary Cell Cultures via CRISPR‐Cas9 Ribonucleoprotein Nucleofection

Abstract

CRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR‐Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2′‐O‐methyl‐3′phosphorothioate‐modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion‐deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient‐derived glioblastoma (GBM) stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem‐like cells. © 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion‐deletion (indel) mutation and genomic deletion generation in primary cell cultures

Mitochondrial Redox Adaptations Enable Aspartate Synthesis in SDH-deficient Cells

“…This protocol was adapted from Hoellerbauer et al, 2020. Three chemically synthesized 2’-O-methyl 3’phosphorothioate-modified single guide RNA (sgRNA) sequences targeting SDHB were purchased (Synthego).…”

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…In addition to use in ssODN, gRNAs have been adapted to be chemically synthesized as 2′- O -methyl-3′-phosphorothioate-modified gRNAs. The phosphorothioate results in an increase in stability and protects against exonucleases, as well as it improves gene editing efficiency of CRISPR/Cas9 to over 90% ( Hendel et al, 2015 ; Hoellerbauer et al, 2020 ). Moreover, phosphorothioate-modified gRNAs have reduced off-target risk compared to the gRNA from plasmid or viral delivery ( Cameron et al, 2017 ).…”

Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens

“…4A). Cell lines encoding the wildtype or mutant proteins were then electroporated with a Cas9 in complex with 1-2 synthetic sgRNAs either targeting the endogenous protein or not targeting the genome at all (Hoellerbauer et al, 2020a;Hoellerbauer et al, 2020b). Doxycycline was either withheld or added after electroporation to test the effect of endogenous gene knockout and whether expression of the wildtype or mutant protein complemented its essential activity.…”

“…Cas9 resistant and mutant alleles were generated as gBlocks (IDT) and inserted into restriction enzyme linearized pcDNA5 FRT/TO/Hygro by Gibson assembly and sequence verified. sgRNA:Cas9 mediated gene knockout Genes were knocked out using 1-2 synthetic sgRNAs (Snythego) in complex with spCas9 (Aldeveron) that were electroporated into cells using a nucleofector system (Lonza) according to published methods (Hoellerbauer et al, 2020a;Hoellerbauer et al, 2020b). Briefly, 120k cells were mixed with either targeting or non-targeting sgRNA:Cas9 complexes in complete SE nucleofector solution.…”

“…All other coding sequences were generated as codon optimized and thus sgRNA resistant gBlocks (IDT) and inserted into restriction enzyme linearized pcDNA5 FRT/TO/Hygro by Gibson assembly and sequence verified. sgRNA:Cas9 mediated gene knockout Genes were knocked out using 1-2 synthetic sgRNAs (Synthego) in complex with spCas9 (Aldeveron 9214) that were electroporated into cells using a nucleofector system (Lonza V4XC-1032) according to published methods (Hoellerbauer et al, 2020a;Hoellerbauer et al, 2020b).…”