Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking (2025)

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges

Current issues in molecular biology, 2018

Precise nucleic acid editing technologies have facilitated the research of cellular function and the development of novel therapeutics, especially the current programmable nucleases-based editing tools, such as the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas). As CRISPR-based therapies are advancing toward human clinical trials, it is important to understand how natural genetic variation in the human population may affect the results of these trials and even patient safety. The development of "base-editing" technique allows the direct, stable transformation of target DNA base into an alternative in a programmable way, without DNA double strand cleavage or a donor template. Genome-editing techniques hold promises for the treatment of genetic disease at the DNA level by blocking the sequences associated with disease from producing disease-causing proteins. Currently, scientists can select the gene they want to modi...

CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing

Scientific reports, 2016

The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded...

Enabling large-scale genome editing by reducing DNA nicking

2019

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Nature Methods, 2015

Articles nAture methods | VOL.12 NO.3 | MARCH 2015 | 237

Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in

Scientific Reports

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use. Reverse genetics, the study of phenotypes due to the targeted mutation of a gene, has been and still is largely used to decipher genes or genetic elements function. Historically, this approach was only possible in model organisms where classical Homologous Recombination (cHR) could introduce genomic alterations in Embryonic Stem (ES) cells or zygotes, i.e. mice 1. Recent advances in gene editing methods with the discovery of ZFNs (Zinc Finger Nucleases), TALENs (Transcription Activator-Like Effector Nucleases), and most recently CRISPR enzymes (Clustered Regularly Interspaced Palindromic Repeats), opened the door to targeted gene modifications in virtually any cell or organism 2. CRISPR is a bacterial acquired immune system involving a DNA cleaving enzyme, such as Cas9, guided by a complementary RNA sequence that can specifically recognize and target invaders' genomic sequences to destroy the invading pathogen 3-5. This system was adapted to mammalian cells to become a programmable gene editing tool and is now largely and commonly used 6-8. CRISPR/Cas9, as well as other nucleases, have the property to induce DNA double strand breaks (DSB) at targeted sequences, resulting in a cellular response to maintain genome integrity. Two repair mechanisms prevail: Non-Homologous End Joining (NHEJ) which produces small insertions or deletions (indels) at the cleavage site, or Homologous Directed Repair that induces the specific insertion of an exogenous DNA fragment at the cut site. Hence, CRISPR/Cas9 has been shown to be efficient for KnockOut (KO) and Knock-In (KI) in cells and zygotes of different species 9-13. It does however show some limitations. Different groups have observed its limited robustness and reproducibility for insertions of large inserts (>1.5 kb), and it has been consistently observed that the CRISPR/Cas9 system in zygotes produces mosaic founders 14,15 , involving further breeding to obtain heterozygous and homozygous animals. Here we performed more than 220 experiments to insert DNA fragments >1.5 kb in more than 120 loci, distributed along the 19 autosomes and X chromosome of mouse ES cells, using either CRISPR/Cas9 or cHR (Sup Table). We systematically used Streptococcus pyogenes (Sp) Cas9 nickase (Cas9n) for all experiments to specifically target the intended insertion site while limiting off-target activity 16-18. While recombined clones could genOway, Lyon, 69007, France.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Journal of Visualized Experiments

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis. Video Link The video component of this article can be found at https://www.jove.com/video/57350/. This quick and inexpensive technology has revolutionized basic research and promises to make a profound impact on the development of personalized disease therapies, precision agriculture, and beyond 2. CRISPR editing is a democratizing tool and implementing the system in a new laboratory requires no particular expertise in genome engineering, just basic molecular biology skills. Researchers can now study previously intractable organisms with a few alternative means for genetic manipulation 3,4. In the past five years alone, CRISPR genome editing has been used to engineer over 200 different vertebrates, invertebrate, plant, and microbial species.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology | NEB

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a longstanding goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

Nature Communications

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

CRISPR/Cas-Dependent and Nuclease-FreeIn VivoTherapeutic Gene Editing

Human Gene Therapy

Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing ex vivo genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) in vivo gene editing. In this review, we provide an overview of the CRISPR/Cas-mediated in vivo genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with in vivo genome editing technologies and strategies used to circumvent them. Despite the robust and precise nucleasemediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.

Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Genome biology, 2018

While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform p...