Improved CRISPR-Cas9 delivery method allows for fast and precise knockouts – Fred Hutch News Service
CRISPR/Cas9 gene-editing technology combined with next-generation sequencing has opened the door for genome-wide screens to identify novel protein functions. CRISPR/Cas9 screens require the delivery of a pool of guide RNAs (gRNA) and Cas9 nuclease into a cell line. The most common and successful delivery approach involves using an “all-in-one” lentiviral vector that encodes the CRISPR/Cas9 system and a library of gRNAs. Because of the pooled nature of the screens, retesting of individual gRNAs is required to validate “hits” from the screen. However, this step has proven challenging for myriad reasons. In lentiviral “all-in-one” systems, gene editing takes several days to occur, and the final pool of cells is often a mixture of wild-type and mutants. For essential genes, the long-windows of gene editing result in a mix of live and dead cells, making it difficult to evaluate their biological effect.
Recent publications in the journals Cancer Reports and Current Protocols in Stem Cell Biology from the Paddison Lab (Human Biology Division) describe an alternative and more efficient approach to CRISPR-Cas9:gRNA delivery that facilitates single gRNA validation in primary cells and cell lines. Molecular and Cellular Biology graduate student, Pia Hoellerbauer, led the study; she explained the significance of their work: “CRISPR-Cas9-based technologies have revolutionized our ability to manipulate the genome, but efficient genome editing in primary cells can still prove challenging. The method we describe here for primary cells is unique in that it is extremely efficient and fast, allowing for over 90% insertion-deletion mutation formation in only three days, even in hyperdiploid cells, such as many cancer cells.”
An effective alternative to lentiviral delivery of the CRISPR-Cas9:gRNA is the delivery of purified Cas9 protein and gRNAs in a ribonucleoprotein complex (RNP), which allows for faster editing because the cell does not need to generate Cas9 protein and the gRNAs from DNA. To build upon this technique, the investigators tested chemically synthesized gRNAs and delivered the complexes through nucleofection. Initially invented by Amaxa (now Lonza), this modified electroporation technique delivers the complexes directly to the nucleus of cultured cells.
They first generated two kinds of CRISPR-Cas9:gRNA RNPs, one with unmodified gRNAs, and the other with modifications in the gRNA known to protect it from degradation by cellular nucleases. Gene editing across multiple patient-derived cell cultures (with different ploidy levels) mediated by RNPs with modified gRNAs yielded superior and faster indel formation at lower doses than RNPs with unmodified gRNAs. Importantly, they observed that most of the editing (>60% indel formation) is completed by 24 hours, and it reaches its peak (>90%) by 72 hours, a significant improvement from lentiviral methods that require up to 12 days to achieve high frequencies of gene-editing.
A high percentage of the CRISPR-Cas9-induced mutations the investigators identified were small insertions and deletions (indels), mostly of one base pair (bp) that are predicted to generate frameshifts resulting in gene knockouts. This finding led them to hypothesize that using two gRNAs in close proximity (50-1000bp) could result in the precise deletion of the entire region between the two gRNA sites. The investigators tested the double gRNA RNP to target several different genes and found a high frequency of precise deletions at the predicted sites. “This means that this method can be used to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. Since this method can be readily applied to many different cell types by varying the nucleofection conditions, we hope that our work encourages other researchers to adopt it for their own applications,” added Hoellerbauer.
Principal investigator Dr. Patrick Paddison explained how this technique could improve screen validations: “This technique is a big help for validating the results of pooled CRISPR-Cas9 screen screens, where thousands of genes are screened simultaneously. Pia’s technique allows us to quickly and uniformly knock out candidate positives from these screens in a few days. Her technique permits small or big deletions, which enables all sorts of manipulations of coding genetic elements.” Their work also showed that this method could easily be used to target multiple genes in the same cell line by consecutive nucleofection of RNPs. Using the successive nucleofection method, the investigators targeted four genes commonly mutated or deleted in adult glioblastoma tumors. Following each editing event, they determined the gene expression changes through RNA-seq followed by differential expression analysis. This application highlights the importance of their method as an approach to model oncogenic transformation.
This work was supported by grants from the National Institutes of Health, American Cancer Society, and by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation
Hoellerbauer, P., Kufeld, M., Arora, S., Wu, H. J., Feldman, H. M., & Paddison, P. J.2020. A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures. Cancer reports (Hoboken, N.J.), ecnr21269. Advance online publication. https://doi.org/10.1002/cnr2.1269
Hoellerbauer, P., Kufeld, M., & Paddison, P. J. 2020. Efficient multi‐allelic genome editing of primary cell cultures via CRISPR‐Cas9 ribonucleoprotein nucleofection. Current Protocols in Stem Cell Biology, 54, e126. doi: 10.1002/cpsc.126
Fred Hutch/UW Cancer Consortium member Patrick Paddison contributed to this work.
Published at Mon, 21 Sep 2020 16:17:48 +0000