scholarly journals Engineered CRISPR-Cas9 nuclease with expanded targeting space

Science ◽  
2018 ◽  
Vol 361 (6408) ◽  
pp. 1259-1262 ◽  
Author(s):  
Hiroshi Nishimasu ◽  
Xi Shi ◽  
Soh Ishiguro ◽  
Linyi Gao ◽  
Seiichi Hirano ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Specific Interaction ◽  
Human Cells ◽  
The Third ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites ◽  
Endogenous Target

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non–base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

scholarly journals A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

2019 ◽  
Author(s):  
Rina C. Sakata ◽  
Soh Ishiguro ◽  
Hideto Mori ◽  
Mamoru Tanaka ◽  
Motoaki Seki ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cytidine Deaminase ◽  
Sequencing Data ◽  
Base Editing ◽  
Dna Molecule ◽  
Deep Sequencing Data ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites ◽  
Sequence Window

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G mutation at target genomic sites, the possible genome editing space when using the current base editors remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of Target-ACE and other base editors were simulated across the human genome, demonstrating the highest potency of Target-ACE to edit amino acid coding patterns. Taking these findings together, Target-ACE is a new tool that broadens the capabilities for base editing for various applications.

scholarly journals Activation-Induced Cytidine Deaminase-Initiated Off-Target DNA Breaks Are Detected and Resolved during S Phase

2012 ◽  
Vol 189 (5) ◽  
pp. 2374-2382 ◽  
Author(s):  
Muneer G. Hasham ◽  
Kathy J. Snow ◽  
Nina M. Donghia ◽  
Jane A. Branca ◽  
Mark D. Lessard ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
S Phase ◽  
Dna Breaks ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

scholarly journals Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

2021 ◽  
Vol 22 (12) ◽  
pp. 6457
Author(s):  
Ho Joung Lee ◽  
Hyun Ju Kim ◽  
Sang Jun Lee
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Target Recognition ◽  
Whole Genome ◽  
Microbial Genomes ◽  
Single Base ◽  
Base Editing ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.

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 The cell cycle restricts activation-induced cytidine deaminase activity to early G1

2016 ◽  
Vol 214 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Qiao Wang ◽  
Kyong-Rim Kieffer-Kwon ◽  
Thiago Y. Oliveira ◽  
Christian T. Mayer ◽  
Kaihui Yao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Nuclear Membrane ◽  
Nuclear Dna ◽  
Time Window ◽  
Cytidine Deaminase ◽  
Dna Lesions ◽  
Cytidine Deaminase Activity ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites

Activation-induced cytidine deaminase (AID) converts cytosine into uracil to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes. In addition, this enzyme produces DNA lesions at off-target sites that lead to mutations and chromosome translocations. However, AID is mostly cytoplasmic, and how and exactly when it accesses nuclear DNA remains enigmatic. Here, we show that AID is transiently in spatial contact with genomic DNA from the time the nuclear membrane breaks down in prometaphase until early G1, when it is actively exported into the cytoplasm. Consistent with this observation, the immunoglobulin (Igh) gene deamination as measured by uracil accumulation occurs primarily in early G1 after chromosomes decondense. Altering the timing of cell cycle–regulated AID nuclear residence increases DNA damage at off-target sites. Thus, the cell cycle–controlled breakdown and reassembly of the nuclear membrane and the restoration of transcription after mitosis constitute an essential time window for AID-induced deamination, and provide a novel DNA damage mechanism restricted to early G1.

scholarly journals CRISPR adenine and cytosine base editors with reduced RNA off-target activities

2019 ◽  
Author(s):  
Julian Grünewald ◽  
Ronghao Zhou ◽  
Sowmya Iyer ◽  
Caleb A. Lareau ◽  
Sara P. Garcia ◽  
...  
Keyword(s):  
Rna Editing ◽  
Cytidine Deaminase ◽  
Single Nucleotide ◽  
Cytidine Deaminases ◽  
Guide Rna ◽  
Adenosine Deaminases ◽  
Dna Base ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Rna

AbstractCRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

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