Abstract Details

E1 - Nonviral Therapeutic Gene Delivery and Synthetic/Molecular Conjugates

338: A Novel High-Throughput Screen to Identify Factors Controlling CRISPR-Mediated Non-Viral Genome Editing

Type: Oral Abstract Session

Presentation Details

Session Title: Nonviral Methods for Delivering Nucleic Acids






Background: After administering a genome editor, the delivery of genome editors is limited by a multi-step process, involving cellular uptake, trafficking, and nuclear import of the vector and its payload. These processes vary widely across cell types and differ depending on the nature of the vector, whether it is a lipid nanoparticle or a different synthetic material. To better understand these delivery limitations within human cells, we developed a novel genome-wide CRISPR screening strategy to identify genes modulating cellular uptake and gene editing efficiency. Methods: Our assay interrogates the cellular processes controlling delivery and stimulating non-homologous end-joining (NHEJ) in human embryonic kidney (HEK293) cells. We transduced HEK293 with a pooled lentivirus sgRNA library comprising of 19,000 target genes (Brunello library), prior to delivering non-viral genome editors. Using a low multiplicity of infection, each of the 19,000 genes in the library was knocked out within a specific subset of cells, allowing us to test the influence of nearly every human gene in a single experiment. To facilitate screening, the lentiviral vector genomically integrated into HEK293 cells included the perturbation identifier (i.e., sgRNA sequences) and the target for NHEJ-based editing within a single PCR-based amplicon (Figure). We delivered Cas9 ribonucleoprotein (RNP) payloads using lipofectamine 2000, to generate double-stranded DNA break within the integrated screening vector (Figure A).Next, we performed next-generation sequencing to quantify indel frequency distribution and sgRNA identity within the same amplicon from genomic DNA collected from the perturbed cell population. The library was sequenced on Illumina NovaSeq 6000 system (paired-end 150 bp reads). Our coverage was >2000 reads per sgRNA in the library. Results: We used MAGeCK analysis to assign ‘Phenotype,' defined as the enrichment of a particular sgRNA in an edited pool of reads. This enrichment phenotype is evidence that the activity of the corresponding gene suppresses NHEJ-mediated Cas9 editing. Based on this enrichment analysis (Figure), we identified 27 top genes involved in trafficking pathways that facilitate enhanced cellular uptake. Upon the knockout of these top genes, the NHEJ editing rate in HEK293 cells increased dramatically. Identified genes were further validated in separate arrayed screen experiments, successfully verifying the reproducibility of our novel screening technique. Knocking out the top hits uncovered in our screen led to a 10-fold increase) in the gene editing efficiency of Cas9 mRNA delivered via lipid-based nanoparticles (e.g., moving from 6% to 60% editing efficiency). Six of the genes also led to a 5-fold increase in Cas9 RNP editing efficiency using lipofectamine delivery. Conclusion: We report a novel genome-wide screen that examines the impact of 19,000 human genes on the delivery and outcomes of genome editors. Distinguished by its potential for large-scale screening, the screen allowed the genome-wide exploration of delivery mechanisms that can be further manipulated for efficient trafficking of CRISPR-Cas9 machinery. We anticipate this high-throughput technique could facilitate the systematic engineering of novel nonviral genome editing delivery strategies.

Plain Language Summary

Our team has made a significant advancement in delivery CRISPR gene editors. We created a new method to study how genes affect the delivery and effectiveness of gene editing tools. Using a special virus to introduce a large set of gene-editing instructions into human kidney cells, we could study the role of nearly 20,000 genes. This approach allowed us to identify 20 genes crucial in how cells receive and integrate these gene-editing tools. When we deactivated these essential genes, we observed a remarkable tenfold increase in the efficiency of the CRISPR-Cas9 gene editing method, using lipid nanoparticles. Our findings open up new possibilities for improving gene editing techniques, especially in cells that do not divide, like those in adult tissues. This could be a big step in treating diseases by correcting genetic errors.

Shivani Saxena¹, Min Zhu², Divya Sinha², Amr Abdeen², David Gamm², Shaoqin Gong², Marcela Martinez², Srikumar Sengupta², Krishanu Saha<sup>2

1</sup>University of Wisconsin-Madison, Middleton, WI,²University of Wisconsin-Madison, Madison, WI"