scholarly journals Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

2019 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Target Site ◽  
Gene Repression ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

scholarly journals A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

2018 ◽  
Author(s):  
Noah Jakimo ◽  
Pranam Chatterjee ◽  
Lisa Nip ◽  
Joseph M Jacobson
Keyword(s):  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Genome Engineering ◽  
Protein Domains ◽  
Human Cells ◽  
Gene Modification ◽  
Editing Efficiency ◽  
Base Editing ◽  
Protospacer Adjacent Motif

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased” nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

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 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 Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Science ◽  
2020 ◽  
Vol 368 (6488) ◽  
pp. 290-296 ◽  
Author(s):  
Russell T. Walton ◽  
Kathleen A. Christie ◽  
Madelynn N. Whittaker ◽  
Benjamin P. Kleinstiver
Keyword(s):  
High Resolution ◽  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Genetic Variants ◽  
Human Cells ◽  
Target Site ◽  
Protospacer Adjacent Motif ◽  
Wide Range ◽  
Almost All ◽  
Site Recognition

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

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 Application of the Modified Cytosine Base-Editing in the Cultured Cells of Bama Minipig

2021 ◽  
Author(s):  
Jia-sheng Pan ◽  
Zi-sheng Lin ◽  
Jian-cong Wen ◽  
Jian-feng Guo ◽  
Xia-hui Wu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Base Pair ◽  
Double Strand Breaks ◽  
Fibroblast Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Base ◽  
Lean Meat ◽  
Base Editing ◽  
Target Effects

Abstract Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

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 Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2099
Author(s):  
Yunxing Liu ◽  
Fang Liang ◽  
Zijiong Dong ◽  
Song Li ◽  
Jianmin Ye ◽  
...  
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Gene Editing ◽  
Zebrafish Genome ◽  
Human Cells ◽  
Ribonucleoprotein Complex ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

scholarly journals Efficient and Accurate Single-Base Genome Editing in Corynebacterium Glutamicum by Target-Mismatched CRISPR/Cpf1

2020 ◽  
Author(s):  
Hyun Ju Kim ◽  
Se Young Oh ◽  
Sang Jun Lee
Keyword(s):  
Corynebacterium Glutamicum ◽  
Genome Editing ◽  
Negative Selection ◽  
Stop Codon ◽  
Single Point ◽  
Point Mutations ◽  
Live Cells ◽  
Single Base ◽  
Base Editing ◽  
Target Dna

Abstract Background CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in C. glutamicum.Results This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in Corynebacterium glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5–95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were accurately edited.Conclusions The present results indicate that the target-mismatched Cpf1 negative selection method is not only efficient in C. glutamicum, but also an accurate single-base genome editing method.

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