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PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.
CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.
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Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA.
Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5'-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17-19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3-5 nucleotide-long 5'-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6-8nt 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.
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A Versatile Tool for the Quantification of CRISPR/Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem/Progenitor Cells.
The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA-DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9-NHEJ, MMEJ, and HDR-and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC.
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Long-Term Rewritable Report and Recording of Environmental Stimuli in Engineered Bacterial Populations.
DNA writing (living sensing recorders) based whole-cell biosensors can capture transient signals and then convert them into readable genomic DNA changes. The primitive signals can be easily obtained by sequencing technology or analysis of protein activity (such as fluorescent protein). However, the functions of the current living sensing recorders still need to be expanded, and the difficulty of rewriting in complex biological environments has further limited their applications. In this study, we designed a long-term rewritable recording system using a CRISPR base editor-based synthetic genetic circuit, named CRISPR-istop. This system can convert stimuli into changes in the fluorescence intensity (reporter) and single-base mutations in genomic DNA (recording). Furthermore, we updated the biological circuit through the strategy of coupling the single-base mutation (record site) and the loss-of-function of the targeted protein (translation stopped by stop codon introduction), and we can remove edited bacteria from a population through selective sweeps upon applying a selective pressure. It successfully conducted the rewritable reporter and recording of the nutrient arabinose and pollutant arsenite with two rounds of continuous operation (10 passages/round, 12 h/passage). These observations indicated that the CRISPR-istop system can report and record stimuli over time; moreover, the recording can be manually erased and rewritten as needed. This method has great potential to be extended to more complicated recording systems to execute sophisticated tasks in inaccessible environments for synthetic biology and biomedical applications, such as monitoring disease-relevant physiological markers or other molecules.
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TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity.
GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). The sensitivity level reached 0.1-10 reads-per-million depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid. TEG-seq was also applied to 22 gRNAs with relatively high in silico ranking score that targeted the biological relevant SNPs. The result indicated the initial selection of gRNAs with high score is important, although it cannot exclude the possibility of off-target.
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Emerging and potential use of CRISPR in human liver disease.
CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.
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In vivo Engineering of Chromosome 19 q-arm by Employing the CRISPR/AsCpf1 and ddAsCpf1 Systems in Human Malignant Gliomas (Hypothesis).
Deletions of the q13.3 region of chromosome 19 have been found commonly in all three main kinds of diffuse human malignant gliomas, powerfully demonstrating the existence of tumor suppressor genes in this region. Consistent with the previous studies, the most common deletion interval has been mapped to a roughly 4 Mb region of 19q13.3 between the APOC2 and HRC genes, between genetic markers D19S219 and D19S246. EML2 is a tumor suppressor gene that is located on 19q13.32 and is considerably methylated in high-grade gliomas. Notably, MIR330 gene that is situated within the non-coding intronic region of EML2 is also detected as an oncosuppressor-miR in a variety of cancers including gliomas. Additionally, glioma oncoprotein Bcl2L12 which is located on 19q13.33 is significantly overexpressed in glioblastoma multiform and has a pivotal role in cancer evolution and resistance to apoptosis. Other genes such as MIR519D and NOP53 are also discovered as tumor suppressor genes in gliomas which are located on 19q13.3 and 19q13.4, respectively. Therefore, we hypothesize that a CRISPR/AsCpf1-based genome engineering strategy might be utilized to attach these deleted sizeable chromosomal portions of genes coding tumor suppressors as vital parts of the chromosome 19 q-arm with the purpose of treatment of this chromosomal abnormality in gliomas. Also, we can concurrently employ the CRISPR-ddAsCpf1 strategy for the precise suppression of Bcl2L12 oncogene in glioma.
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Editing the microbiome the CRISPR way.
Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we discuss how various types of CRISPR-Cas systems can be used to modify the genome of gut microorganisms and bacteriophages. CRISPR-Cas systems can also be delivered to bacterial population and programmed to specifically eliminate members of the microbiome. Finally, engineered CRISPR-Cas systems can be used to control gene expression and modulate the production of metabolites and proteins. Together these tools provide exciting opportunities to investigate the complex interplay between members of the microbiome and our bodies, and present new avenues for the development of drugs that target the microbiome. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.
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Engineering T Cells Using CRISPR/Cas9 for Cancer Therapy.
Recent advances in the development of gene editing technologies, especially the CRISPR/Cas 9 system, have substantially enhanced our ability to make precise and efficient changes in the genomes of various cells. In particular, the genetic engineering of T cells holds huge potential to improve the efficacy and safety of T cells-based cancer therapy. Due to its ease of use and high efficiency, CRISPR/Cas9 enables efficient gene knockout, site-specific knock-in, and genome-wide screen in T cells. Here we review the current progress of applying gene editing to T-cell therapy, focusing on the technical aspects of the CRISPR/Cas9 platform. We also discuss the challenges and future prospects.
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Unraveling therapeutic targets in acute myeloid leukemia through multiplexed genome editing CRISPR screening.
Acute Myeloid Leukemia (AML) is a complex condition driven by genetic changes that disrupt the normal functioning of myeloid cells. Despite progress in treatments, AML prognosis remains challenging, emphasizing the need for innovative therapeutic approaches. CRISPR gene editing, known for its simplicity, efficiency, and cost-effectiveness, surpasses other techniques. The advent of multiplexed CRISPR screens presents intriguing possibilities for exploring AML genetics, unraveling drug resistance mechanisms, and identifying new potential treatment targets.
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