Cas9 Nickase Research Articles

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

Prime editing with genuine Cas9 nickases minimizes unwanted indels

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Addition of the T5 exonuclease increases the prime editing efficiency in plants

Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5′-to-3′ exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dual-pegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.

Cas9-orthologue-mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences.

CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.

The Technologist's Dilemma

The Technologist's Dilemma

PASTE: The Way Forward for Large DNA Insertions.

PASTE: The Way Forward for Large DNA Insertions.

Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety.

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200-350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy. Graphical overview.

Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.

Development of an Inducible Cas9 Nickase and Pam-Free Cas12a Platform for Bacterial Diagnostics

Development of an Inducible Cas9 Nickase and Pam-Free Cas12a Platform for Bacterial Diagnostics

Synthetic Gene Circuit-Based Assay with Multilevel Switch Enables Background-Free and Absolute Quantification of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) detection has found widespread applications in tumor diagnostics and treatment, where the key is to obtain accurate quantification of ctDNA. However, this remains challenging due to the issue of background noise associated with existing assays. In this work, we developed a synthetic gene circuit-based assay with multilevel switch (termed CATCH) for background-free and absolute quantification of ctDNA. The multilevel switch combining a small transcription activating RNA and a toehold switch was designed to simultaneously regulate transcription and translation processes in gene circuit to eliminate background noise. Moreover, such a multilevel switch-based gene circuit was integrated with a Cas9 nickase H840A (Cas9n) recognizer and a molecular beacon reporter to form CATCH for ctDNA detection. The CATCH can be implemented in one-pot reaction at 35 °C with virtually no background noise, and achieve robust absolute quantification of ctDNA when integrated with a digital chip (i.e., digital CATCH). Finally, we validated the clinical capability of CATCH by detecting drug-resistant ctDNA mutations from the plasma of 76 non-small cell lung cancer (NSCLC) patients, showing satisfying clinical sensitivity and specificity. We envision that the simple and robust CATCH would be a powerful tool for next-generation ctDNA detection.

Targeted Mutagenesis in Mice Using a Base Editor.

Base editors, such as cytosine and adenine base editors, are composed of nickase Cas9 (nCas9) and deaminase and serve as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based enzymatic tools for specific nucleotide substitutions. They are mainly the most effective genome editing tools for introducing point mutations, such as C-to-T and A-to-G conversions. The enhanced base editor, a C-to-G base editor (CGBE), can perform other nucleotide substitutions, such as C-to-G conversions. Here, we introduce a method for generating mouse models with point mutations using a base editing system.

TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor

Prime editors (PEs) are powerful tools that widen the possibilities for sequence modifications during genome editing. Although methods based on the analysis of Cas9 nuclease or nickase activity have been used to predict genome-wide off-target activities of PEs, no tool that directly uses PEs for this purpose has been reported yet. In this study, we present a cell-based assay, named TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs. TAPE-seq analyses are successfully performed using many different versions of PEs. The TAPE-seq predictions are compared with results from two other off-site prediction methods, Cas9 nuclease-based GUIDE-seq and Cas9 nickase-based Digenome-seq (nDigenome-seq). TAPE-seq shows a lower miss rate, and a higher area under the receiver operating characteristic curve compared to the other methods. TAPE-seq also identified valid off-target sites that were missed by the other methods.

CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in Shigella flexneri by Cytosine Base Editing.

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted C→T transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. IMPORTANCE Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen-Shigella. This pathogen causes high morbidity and mortality in children, and antibiotic-resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of the utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generate multiple mutants in a single, massively parallel reaction. By virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

288 Traceless, homology-directed repair-mediated gene editing for junctional epidermolysis bullosa

In this study, we used combinations of Cas9 or Cas9 nickase (Cas9n) and single gRNA (sgRNA) ribonucleoprotein (RNP) complexes with single-stranded oligonucleotides (ssODN) to induce homology-directed repair (HDR) in junctional epidermolysis bullosa (JEB) keratinocytes with a pathogenic mutation in exon 52 of COL17A1. To assess the correction efficiency, treated patient keratinocytes were then analysed via next generation sequencing (NGS), Western Blot, sqRT-PCR, Flow cytometry and immunofluorescence stainings of cell monolayers as well as 3D skin equivalents.

RF17 | PMON22 KNOCKOUT AND KNOCKIN GENE EDITING BY CRISPR CAS9 IN ZEBRAFISH (DANIO RERIO) AS A GENOTYPE-PHENOTYPE CORRELATION TOOL FOR EXOME-IDENTIFIED GENES IN PATIENTS WITH CONGENITAL HYPOPITUTARISM

Abstract Introduction Zebrafish has been used as an animal model to study human disease due to its 75% genetic homology with humans besides easier maintenance, small size and high reproductive rate. knockout Gene through CRISPR/Cas genomic edition can lead to an inactivating protein due to frameshift and stop codon, while Knockin allows a specific base edition. Whole exome sequence allows the identification of new/rare variants as molecular diagnosis in patients with congenital hypopituitarism (CH), although sometimes phenotype-genotype is mandatory and edited zebrafish genome can be a tool. The aims of this work was to generate the gh1 knockout gene in zebrafish, mimetizing a patient with CH harboring GH1 c.171delT (p.Phe57Leufs*43) allelic variant leading to frameshift and stop codon and cdh2 knockin due to CDH2 c.865G>A (p.Val289Ile) allelic variant in homozygosity in a patient with CH. Methods For the gh1 knockout gene 2 different guides (sgRNA) were designed in exon 5. At one cell stage a total number of 274 embryos were injected with different sgRNA and Cas9. At 3 months after fertilization, 12 animals had their tail DNA extracted followed by PCR amplification with primers flanking exon 5 and the product was Sanger sequenced. For the cdh2 knockin g>a for p.Val289Ile, base editing plasmid was used, containing an inserted DNA with cytidine deaminase fused to Cas9 nickase (nCas9), that direct convert C to T or G to A in the Zebrafish DNA sequence of interest. Twenty-four hours after injection, a total of 100 embryo's DNA were collected and extracted followed by PCR amplification and Sanger sequenced in order to check mutagenesis efficiency. Results For the gh1 knockout gene animals that reached sexual maturity, two of them with an allelic variant leading to frameshift and stop codon (animal 1 harboring ENSDARG00000038185: g.568_572del, animal 2 harboring ENSDARG00000038185: g.554_560insACGAACG) were prioritized for the strains construction. These animals (F0) were mated with WT animals and their offspring (F1) were genotyped, showing mutations not seen in the tail sequencing of the parents, suggesting a gonadal mosaic in F0 generation. At 3 months these animals (F1) were mated and F2 offspring with pure heterozygous mutation were generated and crossed with WT in order to obtain the mendelian rate of WT, HT and Homo to characterize the animal phenotype. From 100 animals injected for cdh2 knockin approach, 10 random animals have already been sequenced and presented no expected pathogenic allelic variant. Conclusion The genomic editing technique by Crispr cas was efficient to generate a pure heterozygous gh1 knockout animal in order to obtain homozygous animal in F3 for phenotype characterization, while knockin was inefficient for the analyzed number of eggs although it is missing 90 animal to be analyzed in order to calculate the real technique efficiency. Presentation: Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Prime Editing: An Emerging Tool in Cancer Treatment.

Prime Editing is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technique having promising potential in terms of reducing off target activity. It introduces fragments of DNA sequences into the target site using a guide RNA (gRNA) molecule, composed of both the sequence that is to be inserted into the target site along with an inactive Cas9 nickase and a reverse transcriptase. Prime Editing can cause insertions, deletions, and various point mutations for reverting the phenetic characteristics of a disease specially tested in human adult stem cells and cancer cell lines. The main aim of our review is to explore how Prime Editing and its various forms are being utilized as an emerging tool to cure deleterious diseases like cancer, also as a delivery strategy of the tool into cells. There are almost five generations of Prime Editors (PE) with increasing levels of efficiency from one level to another that have huge clinical potential in correcting mutations; however, the necessity for a pegRNA design is extremely significant. But besides having such advantages, the limitations of this technology particularly include generation of double nicks while optimizing the efficiency of PE3. So, it is important to consider all such consequences and customize PE as per requirements.

Construction of a cytidine base editor based on Exopalaemon carinicauda cytidine deaminase and its application in crustacean genome editing

Economic crustaceans play important roles in aquaculture and create great benefits for the world agricultural economy. Studies have found that there are many single nucleotide polymorphism (SNP) sites in the crustacean genomes, which closely related to a variety of life activities like growth, development, reproduction, and so on. However, the in vivo functional study of SNPs still lacks appropriate technique in crustaceans. The cytosine base editor (CBE) based on cytidine deaminase (CDA) can convert the C•G base pair to T•A base pair, which provides a new idea for the study of function of crustacean SNPs. In this study, the CDA gene from Exopalaemon carinicauda (EcCDA) was cloned and fused to the N-terminus of Cas9 nickase (nCas9(D10A)) skeleton to construct a cytidine base editor (pEcCDA-nCas9(D10A)) suitable for crustacean point editing. Neocaridina denticulata sinensis MIH (NdMIH) gene was selected as the target gene. The in vitro transcribed EcCDA-nCas9(D10A) mRNA and NdMIH-gRNA were co-injected into the 1-cell stage embryos of N. denticulata sinensis for gene editing. Detection results showed that the constructed pEcCDA-nCas9(D10A) successfully realized the conversion of C•G to T•A in NdMIH with the editing efficiency of approximately 11.21%. Our works construct a well-established crustacean base editing platform.

FP.42 Correction of point mutations in the DMD gene using the prime editing

Duchenne muscular dystrophy (DMD) is a X-linked hereditary disease responsible for serious burden, pain and damages to patients and their families. About 20 000 children new cases are diagnosed worldwide each year. Existing experimental treatments with antisense oligonucleotides or aminoglycosides provide limited phenotypic improvement. Our study aimed to experiment the CRISPR-Cas9 PRIME editing technology as therapeutic approach. This technology uses a PRIME editor plasmid (PE2) coding for a Moloney murine leukemia virus reverse transcriptase fused with the Cas9 nickase, and a plasmid coding for a prime editing guide RNA (pegRNA) containing in addition to a sgRNA, a primer binding site (PBS) and a reverse transcriptase template (RTT). This system permits specific nucleotide substitutions, deletions or insertions in the genome. We first designed different pegRNAs targeting several human DMD exons (6, 9, 20, 35, 43, 52, 55, 59 and 61) responsible for DMD in Canadian population to introduce a STOP codon by modifying a single nucleotide. We next designed other pegRNAs for the correction of c.428 G>A and c.8713 C>T point mutations respectively in DMD exon 6 and exon 59 in patients' human myoblast. HEK293T and myoblast cells were harvested three to five days post treatment with PE2 and pegRNA plasmids. Parts of myoblasts were transformed into myotubes to obtain proteins for western blot analysis. Fragment of targeted exons were PCR amplified and sequenced by Sanger and Illumina deep sequencing method. Results were analysed using the EditR and CRISPRESSO programs to estimate the editing percentage. We confirmed that PRIME editing permitted the specific C˃T, A˃G, G˃C, G˃A and G˃T substitutions in the DMD gene with an editing efficiency between 3 to 9% and 6 to 21% respectively using PE2 and PE3 approach in HEK293T cells creating point mutations. An additional mutation in the PAM sequence permitted to achieve up to 40% modification. For the correction of point mutations in patients' myoblasts, the modification of RTT length in combination to synonymous additional mutations around the target mutation showed up to 23% correction for a single electroporation with dystrophin expression detected by western blot. Thus Prime editing permits the correction of point mutations in the DMD gene.