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Utilization of CRISPR-Mediated Tools for Studying Functional Genomics in Hematological Malignancies: An Overview on the Current Perspectives, Challenges, and Clinical Implications.
Hematological malignancies (HM) are a group of neoplastic diseases that are usually heterogenous in nature due to the complex underlying genetic aberrations in which collaborating mutations enable cells to evade checkpoints that normally safeguard it against DNA damage and other disruptions of healthy cell growth. Research regarding chromosomal structural rearrangements and alterations, gene mutations, and functionality are currently being carried out to understand the genomics of these abnormalities. It is also becoming more evident that cross talk between the functional changes in transcription and proteins gives the characteristics of the disease although specific mutations may induce unique phenotypes. Functional genomics is vital in this aspect as it measures the complete genetic change in cancerous cells and seeks to integrate the dynamic changes in these networks to elucidate various cancer phenotypes. The advent of CRISPR technology has indeed provided a superfluity of benefits to mankind, as this versatile technology enables DNA editing in the genome. The CRISPR-Cas9 system is a precise genome editing tool, and it has revolutionized methodologies in the field of hematology. Currently, there are various CRISPR systems that are used to perform robust site-specific gene editing to study HM. Furthermore, experimental approaches that are based on CRISPR technology have created promising tools for developing effective hematological therapeutics. Therefore, this review will focus on diverse applications of CRISPR-based gene-editing tools in HM and its potential future trajectory. Collectively, this review will demonstrate the key roles of different CRISPR systems that are being used in HM, and the literature will be a representation of a critical step toward further understanding the biology of HM and the development of potential therapeutic approaches.
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Concepts and tools for gene editing.
Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR-Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.
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Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.
The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.
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The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.
Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.
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Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.
CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.
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Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans.
Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation. This article has an associated First Person interview with the first author of the paper.
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RNA-guided DNA insertion with CRISPR-associated transposases.
CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.
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Accelerated breeding for early-maturing and aromatic glutinous restorer lines with CRISPR/Cas9-mediated targeted editing for hybrid rice.
Hybrid rice breeding depends on the development of elite parental lines with favorable traits such as grain quality, heading time, and plant architecture. However, improving restorer lines through conventional breeding is time-consuming and labor-intensive. Here, we employed a multiplex CRISPR/Cas9 editing strategy to simultaneously target Wx, Badh2, and Se14 in the elite restorer line FH676, aiming to generate glutinous, aromatic, and early-maturing lines. Through Agrobacterium-mediated transformation, we obtained Wx/Badh2 double mutants (Dm) and Wx/Badh2/Se14 triple mutants (Tm). Grain quality analysis revealed significantly reduced amylose content and enhanced aroma content in the edited lines, consistent with Wx and Badh2 knockouts. The triple mutants also exhibited significantly earlier heading compared to the wild type. The early-maturing Tm lines and the Dm lines achieved grain yields of 39.2 ~ 39.4 g and 42.9 ~ 43.0 g per plant, respectively, both exceeding yields of conventional glutinous cultivars used at present. To evaluate hybrid performance, we crossed Tm and Dm lines with two sterile lines: LX (aromatic) and NX (glutinous). The LX/Tm hybrid headed 4.8 days earlier than its wild-type counterpart, with no reduction in plant height or yield. The NX/Tm hybrid showed an advance of ~ 6.3 days in heading but a 6.2 ~ 7.0% reduction in yield due to decreased grain number per panicle. The Se14 knockout likely relieves repression of RFT1 expression during the floral transition under long-day conditions. Variations in phenotypic response across different maternal backgrounds suggest epistatic interactions affecting the dosage response of Se14. In summary, Se14 is a promising target for engineering early-maturing hybrid rice. The edited FH676 lines provide valuable germplasm resource for developing early-maturing, aromatic, and glutinous hybrids through CRISPR-based genome editing.
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Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.
Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5'-DHR-3' PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a "flexible to stable" transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1hf, was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.
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Intrinsic Nucleotide Preference of Diversifying Base Editors Guides Antibody Ex Vivo Affinity Maturation.
Base editors (BEs) are emerging tools used for precision correction or diversifying mutation. It provides a potential way to recreate somatic hypermutations (SHM) for generating high-affinity antibody, which is usually screened from antigen-challenged animal models or synthetic combinatorial libraries. By comparing somatic mutations in the same genomic context, we screened engineered deaminases and CRISPR-deaminase coupling approaches and updated diversifying base editors (DBEs) to generate SHM. The deaminase used in DBEs retains its intrinsic nucleotide preference and mutates cytidines at its preferred motifs. DBE with AID targets the same hotspots as physiological AID does in vivo, while DBE with other deaminases generates distinct mutation profiles from the same DNA substrate. Downstream DNA repair pathways further diversified the sequence, while Cas9-nickase restricted mutation spreading. Finally, application of DBE in an antibody display system achieved antibody affinity maturation ex vivo. Our findings provide insight of DBE working mechanism and an alternative antibody engineering approach.
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