scholarly journals High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity

2021 ◽  
Vol 22 (24) ◽  
pp. 13301
Author(s):  
Dan Zhu ◽  
Junyi Wang ◽  
Di Yang ◽  
Jianzhong Xi ◽  
Juan Li
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
Genome Engineering ◽  
Human Cells ◽  
Wild Type ◽  
Guide Rnas ◽  
Promising Tool ◽  
Target Sites ◽  
Editing Activity ◽  
Type V

CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position −3 for PAM TATV and at position −4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.

scholarly journals Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

2020 ◽  
Author(s):  
Xi Xiang ◽  
Kunli Qu ◽  
Xue Liang ◽  
Xiaoguang Pan ◽  
Jun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
High Efficiency ◽  
Outcome Data ◽  
Massively Parallel ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Sites ◽  
In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

2019 ◽  
Author(s):  
Yan-ru Cui ◽  
Shao-jie Wang ◽  
Jun Chen ◽  
Jie Li ◽  
Wenzhang Chen ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Therapeutic Agent ◽  
Genetic Diseases ◽  
Ectopic Expression ◽  
Human Cells ◽  
Guide Rna ◽  
Editing Activity ◽  
Periplasmic Domain ◽  
Target Activity

AbstractBackgroundCRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

scholarly journals An optimized base editor with efficient C-to-T base editing in zebrafish

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yu Zhao ◽  
Dantong Shang ◽  
Ruhong Ying ◽  
Hanhua Cheng ◽  
Rongjia Zhou
Keyword(s):  
Gene Editing ◽  
Cytidine Deaminase ◽  
Model Organism ◽  
Model Organisms ◽  
Base Substitution ◽  
Base Editing ◽  
Codon Preference ◽  
Promising Tool ◽  
Human Genes ◽  
Target Sites

Abstract Background Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Results Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Conclusions Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

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 with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

2015 ◽  
Author(s):  
Fillip Port ◽  
Nadine Muschalik ◽  
Simon L Bullock
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Basic Research ◽  
Evidence Based ◽  
Gene Drive ◽  
Highly Active ◽  
Genome Modification ◽  
Target Sites ◽  
Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

scholarly journals Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yingxiao Zhang ◽  
Qiurong Ren ◽  
Xu Tang ◽  
Shishi Liu ◽  
Aimee A. Malzahn ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Plant Genome ◽  
Gene Repression ◽  
Wild Type ◽  
Editing Efficiency ◽  
Editing Activity ◽  
Single Transcript ◽  
Genomic Regions ◽  
Crop Genome

AbstractCRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.

scholarly journals A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction

2021 ◽  
Author(s):  
Ofir Yaish ◽  
Maor Asif ◽  
Yaron Orenstein
Keyword(s):  
Data Processing ◽  
Gene Editing ◽  
Target Site ◽  
Systematic Evaluation ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Sites ◽  
Evaluation Of Data

AbstractCRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this gene-editing technique is quite accurate in the target region, there may be many unplanned off-target edited sites. Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of off-target sites) produced by experimental techniques to detect off-target sites with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect off-target sites, was used to produce a dataset of unprecedented scale and quality (more than 200,000 off-target sites over 110 guide RNAs). In addition, the same study included GUIDE-seq experiments for 58 of the guide RNAs to produce in vivo measurements of off-target sites. Here, we fill the gap in previous computational methods by utilizing these data to perform a systematic evaluation of data processing and formulation of the CRISPR off-target site prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive off-target sites to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between the guide RNA and the off-target site as a feature. Finally, we present predictive off-target in vivo models based on transfer learning from in vitro. Our conclusions will be instrumental to any future development of an off-target predictor based on high-throughput datasets.

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.

scholarly journals Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

2018 ◽  
Author(s):  
Josh Tycko ◽  
Luis A. Barrera ◽  
Nicholas Huston ◽  
Ari E. Friedland ◽  
Xuebing Wu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput Screening ◽  
Human Cells ◽  
High Fidelity ◽  
Guide Rnas ◽  
Target Sites ◽  
Library Screen ◽  
Downstream Analysis ◽  
Nucleic Acid Interactions ◽  
Large Repertoire

AbstractWe report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporated randomized barcodes that enable ‘whitelisting’ of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

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