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 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 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 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 Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

2020 ◽  
Vol 2 ◽  
Author(s):  
Limin Hu ◽  
Olalekan Amoo ◽  
Qianqian Liu ◽  
Shengli Cai ◽  
Miaoshan Zhu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Molecular Breeding ◽  
Target Genes ◽  
Agronomic Traits ◽  
Crop Improvement ◽  
Brassica Napus L ◽  
Editing Efficiency ◽  
Base Editing ◽  
Expected Gain ◽  
Genome Wide

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

scholarly journals Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Minh Thuan Nguyen Tran ◽  
Mohd Khairul Nizam Mohd Khalid ◽  
Qi Wang ◽  
Jacqueline K. R. Walker ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Dna ◽  
Dna Editing ◽  
Target Activity ◽  
Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

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 Engineered Campylobacter jejuni Cas9 variant with enhanced activity

2021 ◽  
Author(s):  
Ryoya Nakagawa ◽  
Soh Ishiguro ◽  
Sae Okazaki ◽  
Hideto Mori ◽  
Mamoru Tanaka ◽  
...  
Keyword(s):  
Genome Editing ◽  
Campylobacter Jejuni ◽  
Genome Engineering ◽  
Cytosine Deaminase ◽  
Human Cells ◽  
Adeno Associated Virus ◽  
Cleavage Activity ◽  
Protospacer Adjacent Motif ◽  
Its Gene

Abstract The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

scholarly journals Divergent PAM Specificity of a Highly-Similar SpCas9 Ortholog

2018 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
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. The range of sequences that CRISPR endonucleases can recognize, however, is constrained by the need for a specific protospacer adjacent motif (PAM) flanking the target site. In this study, we demonstrate the natural PAM plasticity of a highly-similar, yet previously uncharacterized, Cas9 fromStreptococcus canis(ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report a divergent affinity to 5’-NNGT-3’ PAM sequences and demonstrate the editing capabilities of the ortholog in both bacterial and human cells. Finally, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise-overlookedStreptococcusCas9 orthologs. Our results establish that ScCas9 can be utilized both as an alternative genome editing tool and as a functional platform to discover novelStreptococcusPAM 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 Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

2019 ◽  
Author(s):  
Wesley A. Wierson ◽  
Brandon W. Simone ◽  
Zachary WareJoncas ◽  
Carla Mann ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Human Cells ◽  
Effector Proteins ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Strand Annealing

AbstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the targeting of DNA double-strand breaks (DSBs) to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ~20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in Protospacer Adjacent Motif (PAM) requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and human cells. Using a fluorescent reporter system, we show that CRISPR/ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR/ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

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