High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

Abstract

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24 hours after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery.

Manufacturing Cell Therapies Using Engineered Biomaterials

“…Techniques such as micropatterning, that segregate populations into unique units, lend themselves well to high content imaging, analysis and screening [69] which enables facile assaying of phenotype and the selection of homogenous subpopulations. Previous studies demonstrated the utility of this in situ monitoring approach in evaluating heterogeneity in CRISPR-Cas9 delivery strategies [70] and editing of pluripotent stem cells [71]. Taken together, this body of work illustrates how biomaterials can provide a powerful platform for the guidance, monitoring, analysis, and quality control of cell populations.…”

“…This can be done using multivariate design of experiments (DOE) approaches to systematically examine the significance and interactions of process parameters, while reducing sample size and effort required compared to conventional methods. For example, integrating biomaterials with DOE methods allow efficient optimization of biomaterial properties for factor delivery[70] or cell functionality[86]. Altogether, biomaterials are suitable as standardized reference materials that could be used as benchmarks in the biomanufacturing process.…”

From the Immune Response to the Genome Design; CRISPR-Cas9 System: Review

“…Bunlarla birlikte ticari olarak bulunan transfeksiyon kitleri de değişen oranlardaki verimlilikle CRISPR-Cas9 sisteminin taşınmasında kullanılmıştır. 59 Non-viral gen aktarımı yöntemleri arasında, fiziksel yöntemler olarak bilinen yön-temler de CRISPR-Cas9 sisteminin hücrelere aktarılmasında kullanılabilir. Bunlar arasında elektroporasyon, mikroinjeksiyon ve hidrodinamik injeksiyon bulunmaktadır.…”

“…51,60,61 Genel olarak viral ve viral olmayan vektörler karşılaştırılacak olursa Tablo 2'deki gibi bir durum ortaya çıkmaktadır. 44,59,62,63 Buna göre viral vektör-ler yüksek verimle CRISPR-Cas9 sisteminin taşın-masında kullanılma potansiyeline sahiptir. Ancak bunun yanında mutajenite ve immunojenite riski taşımalarından dolayı viral olmayan transfeksiyon ve gen taşıma yöntemlerinin önemi ortaya çık-maktadır.…”

Optimizing Crispr Cas9 Genome Editing System:A Review

“…This system is able to create both germ line and somatic animal models with point mutations, deletions and complex chromosomal rearrangements and thus could be directly applied to pain related 49 . Microfluidic membrane deformation method to deliver sgRNA and Cas9 into various cell types such as hard to transfect lymphoma cells and embryonic stem cells while maintaining high cell viability have been developed and have demonstrated to be highly efficient in genome editing and successful in generation of gene-knockout cell lines.…”