The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which genetic diseases and individual disease susceptibilities are both predictable and actionable. Likewise, genes responsible for plant traits can be identified and altered quickly, transforming the pace of agricultural research and plant breeding. In this Review, we discuss the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants along with relevant successes and challenges and present a roadmap for the future of this technology. Description A decade of CRISPR In the decade since the publication of CRISPR-Cas9 as a genome-editing technology, the CRISPR toolbox and its applications have profoundly changed basic and applied biological research. Wang and Doudna now review the origins and utility of CRISPR-based genome editing, the successes and current limitations of the technology, and where innovation and engineering are needed. The authors describe important advances in the development of CRISPR genome-editing technology and make predictions about where the field is headed. They also highlight specific examples in medicine and agriculture that show how CRISPR is already affecting society, with exciting opportunities for the future. —DJ A review discusses the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants and considers its future potential. BACKGROUND The fields of molecular biology, genetics, and genomics are at a critical juncture—a moment in history when a convergence of knowledge and methods has made it both technically possible and incredibly useful to edit specific base pairs or segments of DNA in cells and living organisms. The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which we can not only diagnose human diseases and even predict individual susceptibility based on personal genetics but also act on that information. Likewise, we can both identify and rapidly alter genes responsible for plant traits, transforming the pace of agricultural research and plant breeding. The applications of this technology convergence are profound and far reaching—and they are happening now. In the decade since the publication of CRISPR-Cas9 as a genome editing technology, the CRISPR toolbox and its applications have profoundly changed biological research, impacting not only patients with genetic diseases but also agricultural practices and products. As a specific example from the field of genomic medicine, it has become feasible to obtain a complete sequence of the human genome in less than 24 hours—a staggering advance considering the first such sequence took 5 years to generate. Notably, designing and putting to use a potent CRISPR genome editor to obtain clinically actionable information from that genome—previously a near-intractable challenge—now takes only a matter of days. For additional background and related topics, we refer readers to in-depth reviews of the microbiology and structural biology of CRISPR systems and to articles about the considerable ethical and societal challenges of this technology. ADVANCES The past decade has witnessed the discovery, engineering, and deployment of RNA-programmed genome editors across many applications. By leveraging CRISPR-Cas9’s most fundamental activity to create a targeted genetic disruption in a gene or gene regulatory element, scientists have built successful platforms for the rapid creation of knockout mice and other animal models, genetic screening, and multiplexed editing. Beyond traditional CRISPR-Cas9–induced knockouts, base editing—a technology utilizing engineered Cas9’s fused to enzymes that alter the chemical nature of DNA bases—has also provided a highly useful strategy to generate site-specific and precise point mutations. Over the past decade, scientists have utilized CRISPR technology as a readily adaptable tool to probe biological function, dissect genetic interactions, and inform strategies to combat human diseases and engineer crops. This Review covers the origins and successes of CRISPR-based genome editing and discusses the most pressing challenges, which include improving editing accuracy and precision, implementing strategies for precise programmable genetic sequence insertions, improving targeted delivery of CRISPR editors, and increasing access and affordability. We examine current efforts addressing these challenges, including emerging gene insertion technologies and new delivery modalities, and describe where further innovation and engineering are needed. CRISPR genome editors are already being deployed in medicine and agriculture, and this Review highlights key examples, including a CRISPR-based therapy treating sickle cell disease, a more nutritious CRISPR-edited tomato, and a high-yield, disease-resistant CRISPR-edited wheat, to illustrate CRISPR’s current and potential future impacts in society. OUTLOOK In the decade ahead, genome editing research and applications will continue to expand and will intersect with advances in technologies, such as machine learning, live cell imaging, and sequencing. A combination of discovery and engineering will diversify and refine the CRISPR toolbox to combat current challenges and enable more wide-ranging applications in both fundamental and applied research. Just as during the advent of CRISPR genome editing, a combination of scientific curiosity and the desire to benefit society will drive the next decade of innovation in CRISPR technology. CRISPR: past, present, and future. The past decade of CRISPR technology has focused on building the platforms for generating gene knockouts, creating knockout mice and other animal models, genetic screening, and multiplexed editing. CRISPR’s applications in medicine and agriculture are already beginning and will serve as the focus for the next decade as society’s demands drive further innovation in CRISPR technology.

CRISPR technology: A decade of genome editing is only the beginning

Prime editing for precise and highly versatile genome manipulation

Advancing CAR T cell therapy through the use of multidimensional omics data

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides the best balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

Carbon-negative synthetic biology: challenges and emerging trends of cyanobacterial technology.

High-content CRISPR screening

Reversing HIV-1 latency promotes killing of infected cells and is essential for cure strategies; however, no single latency reversing agent (LRA) or LRA combination have been shown to reduce HIV-1 latent reservoir size in persons living with HIV-1 (PLWH). Here, we describe an approach to systematically identify LRA combinations to reactivate latent HIV-1 using genome-wide CRISPR screens. Screens on cells treated with suboptimal concentrations of an LRA can identify host genes whose knockout enhances viral gene expression. Therefore, inhibitors of these genes should synergize with the LRA. We tested this approach using AZD5582, an activator of the noncanonical nuclear factor κB (ncNF-κB) pathway, as an LRA and identified histone deacetylase 2 (HDAC2) and bromodomain-containing protein 2 (BRD2), part of the bromodomain and extra-terminal motif (BET) protein family targeted by BET inhibitors, as potential targets. Using CD4+ T cells from PLWH, we confirmed synergy between AZD5582 and several HDAC inhibitors and between AZD5582 and the BET inhibitor, JQ1. A reciprocal screen using suboptimal concentrations of an HDAC inhibitor as an LRA identified BRD2 and ncNF-κB regulators, especially BIRC2, as synergistic candidates for use in combination with HDAC inhibition. Moreover, we identified and validated additional synergistic drug candidates in latency cell line cells and primary lymphocytes isolated from PLWH. Specifically, the knockout of genes encoding CYLD or YPEL5 displayed synergy with existing LRAs in inducing HIV mRNAs. Our study provides insights into the roles of host factors in HIV-1 reactivation and validates a system for identifying drug combinations for HIV-1 latency reversal. Description A genome-wide screen for drugs targeting the latent HIV-1 reservoir reveals candidate latency reversing agents. Screen time Curing HIV-1 will require the ability to reverse latency of the virus, but effective latency reversal agents (LRAs) have yet to be identified. Here, Dai et al. developed a genome-wide CRISPR screen to identify targets that promote HIV-1 latency reversal. Using the screen, the authors both validated existing LRAs and identified genes that merit investigation as LRA targets, such as CYLD and YPEL5. Knockout of these genes synergized with existing LRAs, suggesting that combinatorial approaches may be the best path forward for inducing latency reversal.

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir


 Functional genetic screens have been frequently used to repurpose existing drugs or to identify new drug targets. However, the associations between hits on screens and candidate drugs have been unclear, and a manual approach to directly infer drug candidates from “druggable” candidates suffers from its laborious, ad hoc investigation and low accuracy. Here, we describe a systematic approach to explore the associations between genetic dependencies and drug sensitivity of cancer cell lines and to identify potential drug repositioning opportunities. We first developed drug sensitivity predictive models trained on genome-wide CRISPR-Cas9 knockout viability profiles and large-scale drug response screens of 4,518 drugs from DepMap. We obtained the highest predictive power from the decision-tree-based models and observed previously reported associations between gene perturbation and drug response. For example, cancer cells that are more sensitive to TP53 or MDM2 perturbation were more sensitive to MDM inhibitors, and EGFR or ERBB2 dependency strongly predicted the sensitivity of cells to EGFR inhibitors. Interestingly, for 3,260 drugs with known targets, only 1.5% of these genes were selected as top features to predict drug sensitivity, indicating that genes that are not the direct targets of drugs should be considered. In addition, we applied our models to the CRISPR-Cas9 knockout screens that target 1,805 “druggable” genes and predicted the sensitivity of cancer cells to known and novel drugs. Furthermore, we were able to validate the candidate drugs using cell viability assays. Finally, a multi-class classification deep learning model, based on the TorchDrug framework, reaches higher recall values than a decision-tree-based method but with much lower precision values. Our study provided the first systematic evaluation between large-scale gene dependency and drug responses. The machine learning predictive model provides an in-silico approach to perform drug screens from functional genetic screens.
 Citation Format: Bicna Song, Lumen Chao, Xiaolong Cheng, Yuan Gao, Ruocheng Shan, Wei Li. Predictive modeling of drug sensitivity based on the genetic dependency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB069.

Bicna Song

Papers

High-content CRISPR screening

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir

Abstract LB069: Predictive modeling of drug sensitivity based on the genetic dependency