Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering

Bloemberg, Darin; Sosa-Miranda, Carmen Daniela; Nguyen, Tina; Weeratna, Risini D.; McComb, Scott

doi:10.1089/crispr.2020.0090

Cited by 3 publications

(2 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Transfection of Cas9 and gRNA encoding vectors as well as donor plasmids with fluorescent selectable markers lead to integration of the donor and constitutive expression of double-positive fluorescent cells. Coinciding with previous findings ( 54 , 62 , 75 ), we were able to observe 1-2% double positive cells, depending on the cell type and gRNAs used. Performing one sorting step using FACS, we were able to create populations consisting of up to 97.6% constitutively double-positive cells in cell lines and 72.0% in ASCs.…”

Section: Discussionsupporting

confidence: 90%

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication

et al. 2022

View full text Add to dashboard Cite

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

show abstract

Section: Discussionsupporting

confidence: 90%

Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication

et al. 2022

View full text Add to dashboard Cite

show abstract

“…These variants exhibit improved fidelity compared to SpCas9 and can substantially reduce the occurrence of off-target effects. Once the spCas9 variant is generated, the subsequent challenge involves selecting the most suitable variant for a specific target sequence [192] that can also inactivate the Cas9 protein promptly. Replacing plasmids with RNPs not only ensures higher specificity but also avoids the problem of continuous Cas9 protein expression.…”

Section: Challenges and Perspectivesmentioning

confidence: 99%