High-throughput CRISPR technology: a novel horizon for solid organ transplantation

Abstract

Organ transplantation is the gold standard therapy for end-stage organ failure. However, the shortage of available grafts and long-term graft dysfunction remain the primary barriers to organ transplantation. Exploring approaches to solve these issues is urgent, and CRISPR/Cas9-based transcriptome editing provides one potential solution. Furthermore, combining CRISPR/Cas9-based gene editing with an ex vivo organ perfusion system would enable pre-implantation transcriptome editing of grafts. How to determine effective intervention targets becomes a new problem. Fortunately, the advent of high-throughput CRISPR screening has dramatically accelerated the effective targets. This review summarizes the current advancements, utilization, and workflow of CRISPR screening in various immune and non-immune cells. It also discusses the ongoing applications of CRISPR/Cas-based gene editing in transplantation and the prospective applications of CRISPR screening in solid organ transplantation.

Keywords: CRISPR screening; CRISPR/Cas9; high-throughput; solid organ transplantation; transcriptome editing.

Figure 1

Schematic of the CRISPR-Cas system of the natural prokaryotic immune system. After viral invasion, certain bacteria can incorporate a viral gene fragment into their CRISPR storage space. When reencountering the virus, bacteria initiate CRISPR transcription to produce pre-crRNA, which are then processed to create crRNAs. Upon recognizing sequences homologous to the viral gene, the crRNAs guide Cas protein to bind to and cleave the target gene, protecting the bacterial host from viral infiltration. Created with BioRender.com.

Figure 2

Schematic of different CRISPR-Cas system types. (A) A gRNA is used to identify the target genome sequence, guiding the Cas9 endonuclease to precisely cut the DNA double-strand. (B) For gene repression, dCas9 can be fused to KRAB, DNMT3A, or DNMT3A-DNMT3L. (C) Various transcription activators can be fused to dCas9, including VP64, VP192, SunTag arrays, and the VPR comprising VP64, p65, and Rta. (D, E) Combining dCas9 or nCas9 with cytidine or adenine deaminases makes it possible to directly convert cytidine into uridine or adenine into inosine. (F) As a fusion protein, prime editor is composed of reverse transcriptase and nCas9. Prime editor binds to a specific target DNA sequence under the guidance of prime-editing guide RNA (pegRNA). And a single-strand break is made by the nCas9. There are two main components of pegRNA: the reverse transcription template (RTT) and the protospacer binding sequence (PBS). During reverse transcription, the RTT encodes the desired edits which are primed by PBS and the edits are incorporated into the newly synthesized DNA strand. After DNA repair, the edits are stably incorporated into the genome. Created with BioRender.com.

Figure 3

The general workflow of CRISPR screening. Firstly, three or more sgRNAs are designed for each gene, sgRNAs are synthesized with high throughput and cloned into a lentiviral vector. And, the lentiviral library is transduced into target cells and the transduced cells are sorted for sequencing with multiple selective strategies. Finally, the potential genes can be validated with bioinformatic analysis. Created with BioRender.com.

Figure 4

The target genes of CRISPR screening performed in human-derived immune cells discussed in this review. These genes are classified into different research areas, including CAR T therapy, HIV infection, macrophage-mediated disease, and T cell biology. Created with BioRender.com.

Figure 5

The overview of CRISPR screening in solid organ transplantation. Firstly, we should have a scientific question. Subsequently, an appropriate model can be chosen based on the scientific question. And, an optimized gRNA library (custom or genome-wide) should be designed, followed by pooled sgRNA synthesis, plasmid cloning, and viral particle production. Additionally, after transducing the gRNA library into the target cells, they can be sorted for sequencing based on positive or negative selection or different phenotypes. The genomic DNA (gDNA), the global genetic information of sorted cells, need to be isolated and amplified for further analysis. Finally, the potential targets in the donor grafts and immunotherapy can be intervened with the CRISPR/Cas 9 based genome editing, giving the novel insights for clinical problems. Created with BioRender.com.