scholarly journals PAM recognition by miniature CRISPR nucleases triggers programmable double-stranded DNA target cleavage

2019 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Greta Bigelyte ◽  
Joshua K. Young ◽  
Zhenglin Hou ◽  
Rimante Zedaveinyte ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dependent Manner ◽  
Short Sequence ◽  
E Coli ◽  
Defense Systems ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Type V ◽  
Programmable Nucleases ◽  
Cas Proteins

ABSTRACTIn recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas nucleases that recognize and cleave dsDNA in PAM dependent manner. Categorized as class 2 type V-F they come from the Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5’ T- or C-rich PAM sequence triggers double stranded (ds) DNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in E. coli and find that some but not all can. Altogether, our findings show that miniature Cas nucleases are functional CRISPR-Cas defense systems and have the potential to be harnessed as programmable nucleases for genome editing.

scholarly journals PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage

2020 ◽  
Vol 48 (9) ◽  
pp. 5016-5023 ◽  
Author(s):  
Tautvydas Karvelis ◽  
Greta Bigelyte ◽  
Joshua K Young ◽  
Zhenglin Hou ◽  
Rimante Zedaveinyte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Genome Editing ◽  
Dependent Manner ◽  
Short Sequence ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Type V ◽  
Programmable Nucleases ◽  
Cas Proteins

Abstract In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422–603 amino acids) CRISPR–Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5′ T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

2018 ◽  
Author(s):  
Bin Li ◽  
Chunxi Zeng ◽  
Wenqing Li ◽  
Xinfu Zhang ◽  
Xiao Luo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adaptive Immune System ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Modified Dna ◽  
Target Dna ◽  
Editing Activity ◽  
Dose Dependent

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

scholarly journals Biochemically diverse CRISPR-Cas9 orthologs

2020 ◽  
Author(s):  
Giedrius Gasiunas ◽  
Joshua K. Young ◽  
Tautvydas Karvelis ◽  
Darius Kazlauskas ◽  
Tomas Urbaitis ◽  
...  
Keyword(s):  
Temperature Dependence ◽  
Genome Editing ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Programmable Nucleases ◽  
Computational Analyses ◽  
Sequence Requirements ◽  
Insight Into ◽  
Biochemical Diversity

ABSTRACTCRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79 Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements. PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

scholarly journals Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing

2017 ◽  
Author(s):  
Matthew B. Begemann ◽  
Benjamin N. Gray ◽  
Emma January ◽  
Anna Singer ◽  
Dylan C. Kesler ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Genome Editing ◽  
Bacterial Species ◽  
Crop Plant ◽  
Effector Protein ◽  
Enabling Technology ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Type V

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

scholarly journals CRISPR/Cas “non-target” sites inhibit on-target cutting rates

2020 ◽  
Author(s):  
Eirik A. Moreb ◽  
Mitchell Hutmacher ◽  
Michael D. Lynch
Keyword(s):  
Genome Editing ◽  
Target Site ◽  
High Fidelity ◽  
List Type ◽  
Dependent Manner ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Dose Dependent ◽  
Target Activity

AbstractCRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA. The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.HighlightsThe pool of non-target PAM sites inhibit Cas9/gRNA on-target activitynon-target PAM inhibition is dose dependentnon-target PAM inhibition is a function of gRNA sequencenon-target PAM inhibition is a function of Cas9 levels

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3570
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

2016 ◽  
Author(s):  
Bernd Zetsche ◽  
Matthias Heidenreich ◽  
Prarthana Mohanraju ◽  
Iana Fedorova ◽  
Jeroen Kneppers ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Brain ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Simple Route ◽  
Defense Systems ◽  
Crispr System ◽  
Crispr Array ◽  
Type V

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.

scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Joshua C Cofsky ◽  
Deepti Karandur ◽  
Carolyn J Huang ◽  
Isaac P Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Active Site ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Loop Structure ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Type V ◽  
Dna Substrate

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

scholarly journals Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases

2020 ◽  
Vol 48 (7) ◽  
pp. 3722-3733 ◽  
Author(s):  
Eszter Tóth ◽  
Éva Varga ◽  
Péter István Kulcsár ◽  
Virág Kocsis-Jutka ◽  
Sarah Laura Krausz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Plant Cells ◽  
Dependent Manner ◽  
Protospacer Adjacent Motif

Abstract The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

Sign in / Sign up

Export Citation Format

Share Document