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.

scholarly journals Identification and characterization of Fep15, a new selenocysteine-containing member of the Sep15 protein family

2006 ◽  
Vol 394 (3) ◽  
pp. 575-579 ◽  
Author(s):  
Sergey V. Novoselov ◽  
Deame Hua ◽  
Alexey V. Lobanov ◽  
Vadim N. Gladyshev
Keyword(s):  
Mammalian Cells ◽  
Insertion Sequence ◽  
Phylogenetic Analyses ◽  
Dependent Manner ◽  
Putative Active Site ◽  
A Genome ◽  
Sequence Elements ◽  
Identification And Characterization ◽  
Insertion Sequence Elements

Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication.

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 Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility

2020 ◽  
Vol 2 ◽  
Author(s):  
Chengwei Zhang ◽  
Guiting Kang ◽  
Xinxiang Liu ◽  
Si Zhao ◽  
Shuang Yuan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plant Species ◽  
Streptococcus Pyogenes ◽  
Genome Engineering ◽  
Human Cells ◽  
Rice Plants ◽  
Potential Target ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Related Plant

The CRISPR-Cas9 system enables simple, rapid, and effective genome editing in many species. Nevertheless, the requirement of an NGG protospacer adjacent motif (PAM) for the widely used canonical Streptococcus pyogenes Cas9 (SpCas9) limits the potential target sites. The xCas9, an engineered SpCas9 variant, was developed to broaden the PAM compatibility to NG, GAA, and GAT PAMs in human cells. However, no knockout rice plants were generated for GAA PAM sites, and only one edited target with a GAT PAM was reported. In this study, we used tRNA and enhanced sgRNA (esgRNA) to develop an efficient CRISPR-xCas9 genome editing system able to mutate genes at NG, GAA, GAT, and even GAG PAM sites in rice. We also developed the corresponding xCas9-based cytosine base editor (CBE) that can edit the NG and GA PAM sites. These new editing tools will be useful for future rice research or breeding, and may also be applicable for other related plant species.

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

Functional characterization of the mouse Serpina1 paralog DOM-7

2018 ◽  
Vol 399 (6) ◽  
pp. 577-582 ◽  
Author(s):  
Karen Jülicher ◽  
Annabell Wähner ◽  
Kerstin Haase ◽  
Karen W. Barbour ◽  
Franklin G. Berger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Models ◽  
Mouse Strain ◽  
Neutrophil Elastase ◽  
Genome Engineering ◽  
Functional Characterization ◽  
Cdna Clones ◽  
High Complexity ◽  
A1at Deficiency

Abstract The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

scholarly journals Programmable downsizing of CRISPR-Cas9 activity for precise and safe genome editing

2020 ◽  
Author(s):  
Masaki Kawamata ◽  
Hiroshi I. Suzuki ◽  
Ryota Kimura ◽  
Atsushi Suzuki
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Es Cells ◽  
Genomic Alteration ◽  
Dependent Manner ◽  
Simple Modification ◽  
Disease Mutations ◽  
Allele Dosage ◽  
Target Activity

AbstractCRSIPR-Cas9 system has opened up the avenue to efficient genome editing1–4. However, together with known off-target effects, several concerns of current CRISPR-Cas9 platform, including severe DNA damage, cytotoxicity, and large genomic alteration, have emerged in recent reports5–7 and establish a formidable obstacle to precisely model allele dosage effects of disease mutations and risk variants, especially mono-allelic effects, and correct them. Here, by developing an allele-specific indel monitor system (AIMS), we demonstrate that small and simple modification of conventional single-guide RNAs (sgRNAs) enable programmable tuning of CRISPR-Cas9 activities and alleviate such adverse effects. AIMS, which visualizes various indel events in two alleles separately in living cells, is convenient and accurate to determine the in vitro editing efficiency and revealed frequent mosaicism during genome editing. Using AIMS, we show that adding cytosine stretches to the 5’ end of conventional sgRNA efficiently reduced Cas9 activity in a length dependent manner. By combining systematic experiments and computational modeling, we established the quantitative relationships between the length of cytosine extension and multiple aspects of CRISPR-Cas9 system. In general, short cytosine extension dramatically relieves p53-dependent cytotoxicity and suppression of homology-directed repair (HDR) while relatively maintaining on-target activity. Long cytosine extension further decreases on-target activity, thereby maximizing mono-allelic editing, while conventional system typically induces bi-allelic editing. Furthermore, such downregulation of on-target activity contributes to downregulation of relative off-target activity and protection of HDR-allele from second off-target editing. Therefore, cytosine extension method finally enables both single-step generation of heterozygous single-nucleotide disease mutations from homozygous states in mouse ES cells and correction of heterozygous disease mutations in human iPS cells. Taken together, our study proposes updates of standard CRISPR-Cas9 platform in mammalian cells toward precise and safe genome editing in diverse applications.

scholarly journals Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

2019 ◽  
Author(s):  
Zhuqing Wang ◽  
Yue Wang ◽  
Shawn Wang ◽  
Andrew J Gorzalski ◽  
Hayden McSwiggin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Editing Efficiency ◽  
Protospacer Adjacent Motif ◽  
Summary Statement ◽  
Strict Requirement ◽  
Moraxella Bovoculi ◽  
Target Effects

AbstractDespite many advantages over Cas9, Cas12a has not been widely used in genome editing in mammalian cells largely due to its strict requirement of the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM sequences and with minimal on-target mutations and close to 100% editing efficiency when crRNAs of 23nt spacers were used.Summary statementCRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and high overall knock-in efficiency.

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals Minimal PAM specificity of a highly similar SpCas9 ortholog

2018 ◽  
Vol 4 (10) ◽  
pp. eaau0766 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Sequence Space ◽  
Genome Engineering ◽  
Human Cells ◽  
Target Site ◽  
Bioinformatics Pipeline ◽  
Protospacer Adjacent Motif ◽  
Streptococcus Canis

RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5′-NNG-3′ PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.

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.

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