Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases

In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases.

Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

Chloroplast and mitochondrial DNA editing in plants

Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.

Evolutionary Comparative Analyses of DNA-Editing Enzymes of the Immune System: From 5-Dimensional Description of Protein Structures to Immunological Insights and Applications to Protein Engineering

The immune system is unique among all biological sub-systems in its usage of DNA-editing enzymes to introduce targeted gene mutations and double-strand DNA breaks to diversify antigen receptor genes and combat viral infections. These processes, initiated by specific DNA-editing enzymes, often result in mistargeted induction of genome lesions that initiate and drive cancers. Like other molecules involved in human health and disease, the DNA-editing enzymes of the immune system have been intensively studied in humans and mice, with little attention paid (< 1% of published studies) to the same enzymes in evolutionarily distant species. Here, we present a systematic review of the literature on the characterization of one such DNA-editing enzyme, activation-induced cytidine deaminase (AID), from an evolutionary comparative perspective. The central thesis of this review is that although the evolutionary comparative approach represents a minuscule fraction of published works on this and other DNA-editing enzymes, this approach has made significant impacts across the fields of structural biology, immunology, and cancer research. Using AID as an example, we highlight the value of the evolutionary comparative approach in discoveries already made, and in the context of emerging directions in immunology and protein engineering. We introduce the concept of 5-dimensional (5D) description of protein structures, a more nuanced view of a structure that is made possible by evolutionary comparative studies. In this higher dimensional view of a protein’s structure, the classical 3-dimensional (3D) structure is integrated in the context of real-time conformations and evolutionary time shifts (4th dimension) and the relevance of these dynamics to its biological function (5th dimension).

Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

RNA silencing by CRISPR in plants does not require Cas13

RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation and expanded function including anti-viral immunity. Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we discovered that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. We determined that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins or long double-stranded RNA. These results suggest that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement. Our results demonstrate that GIGS is active across a range of plant species, evidence similar to recent findings in an insect system, which suggests that GIGS is potentially active across many eukaryotes.

An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. To better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 39 published data sets. We find strong evidence that the genomic context, which can be defined as the DNA content outside of the gRNA/target sequence itself, greatly contributes to differences in gRNA dependent activity. Context underlies variation in activity often attributed to differences in gRNA sequence. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

From the discovery of DNA to current tools for DNA editing

In 1944, the Journal of Experimental Medicine published the groundbreaking discovery that DNA is the molecule holding genetic information (1944. J. Exp. Med.https://doi.org/10.1084/jem.79.2.137). This seminal finding was the genesis of molecular biology and the beginning of an incredible journey to understand, read, and manipulate the genetic code.

Zp4 is completely dispensable for fertility in female rats†

Zona pellucida (ZP), which is composed of at most four extracellular glycoproteins (ZP1, ZP2, ZP3, and ZP4) in mammals, shelters the oocytes and is vital in female fertility. Several studies have identified the indispensable roles of ZP1–3 in maintaining normal female fertility. However, the understanding of ZP4 is still very poor because only one study on ZP4-associated infertility performed in rabbits has been reported up to date. Here we investigated the function of mammalian Zp4 by creating a knockout (KO) rat strain (Zp4−/− rat) using CRISPR-Cas9 mediated DNA-editing method. The influence of Zp4 KO on ZP morphology and some pivotal processes of reproduction, including oogenesis, ovulation, fertilization and pup production, was studied using periodic acid–Schiff’s staining, superovulation, in vitro fertilization, and natural mating. The ZP morphology in Zp4−/− rats was normal and none of these pivotal processes was affected. This study renewed the knowledge of mammalian Zp4 by suggesting that Zp4 was completely dispensable for female fertility.