Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.
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ABSTRACT:
SUBMITTER: Bak SY
PROVIDER: S-EPMC8565309 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in "seed" sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Strikingly, a subset of variants which broke homology in the intervening region consistently increased the capacity of Cas9 to cleave in extended reactions. Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. Taken together, these studies provide both a basis for predicting effective cleavage targets and a basis for potential optimization of guide RNAs to yield efficiency beyond that of the simple perfect-match guides. 118 samples anaylzed. Controls have con in sample name. To quantitatively measure cleavage efficiency of a single gRNA, we created a population of random variant target sequences to two gRNA targets. The targets used were "unc-22A", [a sequence from the well-characterized unc-22 gene of Caenorhabditis elegans], and "protospacer 4" (ps4), a previously characterized sequence from a natural spacer from S. pyogenes MGAS10750 . Using custom mixtures of oligonucleotide precursors for each base during chemical synthesis, a set of polymorphic target libraries ('Random Variant Libraries') were designed to have a baseline variation rate at each position. On each side of the gRNA homology and PAM regions, 6 bps of random sequence were added. The first base of intended gRNA homology is designated base 1 . The entire 35 bp random variant library mixture was cloned into a standard plasmid vector (pHRL-TK). Several thousand colonies from plates were washed in pools and prepared by standard plasmid preparation methods. The complexity of the libraries were estimated based on Illumina sequencing of the uncut libraries and filtering for minimum representation expected from the pooling. Approximately 1500-3000 unique species were obtained in the unc-22A libraries and 5000 unique sequences in the ps4 library (see Materials and Methods). To assay cleavage, purified Cas9 was first incubated with gRNA, followed by incubation with the variant library for various time points and under various conditions. DNA template is among the conditions varied in the experiments. After protein removal, flanking sequences outside of the target region are used for PCR amplification and plasmid cleavage was measured through loss of PCR products that span the region of interest. A set of perfectly matched targets and highly mutated versions present in the random variant library served as internal positive and negative controls respectively. A log retention score for each sequence in each experiment was calculated by quantifying the representation of each sequence before and after addition of the Cas9 protein. Two approaches were used for normalization: first we used a population of ps4 targets "spiked" into the library as an uncleaved control, second, we used a population of unc-22A targets with large numbers of variations from the perfect target (between 4 and 7), and hence likely limited if any cleavage. Equivalent results are obtained with these two normalization approaches (see Computational Methods for details). Retention scores are expressed as the log2 of the normalized ratio, so that a more negative retention score indicates efficient cleavage of substrate while a less negative score indicates less cleavage. Templates which are uncleaved will yield a retention score at or near zero. Comparisons between multiple experiments indicate strong correlation between independent retention measurements. GSM1410678-GSM1410761; AF_SOL*.dat' files contain the calculated final retentions for each experiment. Each experiment labeled: M-bM-^@M-^\AF_SOL_###_t###M-bM-^@M-^]. M-bM-^@M-^\AF_SOL_###M-bM-^@M-^] corresponds to the experiment run ID and M-bM-^@M-^\t###M-bM-^@M-^] corresponds to the incubation time of the experiment. For example AF_SOL_513_t360, corresponds to experiment 513 on the protospacer 4 guide and DNA target and the incubation time was 360 mins. The experimental conditions and ID can be found in the associated publication. GSM1544297-GSM1544332; unc*.dat file is a tab-delimited file of all considered sequences in each experiment. The names of the files and the AF_SOL_# run number can be found in the associated publication (Supplementary Materials) with the details of the conditions. Each filename starts with the type of gRNA used (either unc-22WT or the mutant version unc22C11G). The next number (#min) is indication of the time of incubation for the experiment and this is either followed by #pcr_AF_SOL_# or just AF_SOL_#. If followed by #pcr, that is the indication of the number of PCR cycles used in the experiments. Finally, AF_SOL_# denotes the sequencing run ID number.
2014-11-14 | E-GEOD-58426 | biostudies-arrayexpress
Project description:The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
| S-EPMC4714946 | biostudies-literature
Project description:Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.
| S-EPMC8053117 | biostudies-literature
Project description:EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals.
| S-EPMC3149191 | biostudies-literature
Project description:To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in "seed" sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Strikingly, a subset of variants which broke homology in the intervening region consistently increased the capacity of Cas9 to cleave in extended reactions. Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. Taken together, these studies provide both a basis for predicting effective cleavage targets and a basis for potential optimization of guide RNAs to yield efficiency beyond that of the simple perfect-match guides.
2014-11-14 | GSE58426 | GEO
Project description:The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.
| S-EPMC4338555 | biostudies-literature
Project description:The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.
| S-EPMC7002122 | biostudies-literature
Project description:RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.
| S-EPMC6800346 | biostudies-literature
Project description:Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.
| S-EPMC4402137 | biostudies-literature
Project description:The specificity of STAT1 transcription factor was determined quantitatively by measuring the relative binding affinity to hundreds of variants of the gonsensus site. The binding specities of eight mutants of STAT1 were also investigated. The mutants showed either overall reduced binding specificity or altered specificity accross the binding site. The effect of CpG methylaton within the binding site was also investigated, which demonstrated verry little effect on binding. A previously described method, Spec-Seq, was used to determine the binding specificititys of STAT1 and the mutants. Libreries of DNAs were enzymatically methylated and the same method was used for studying their binding affinities in comparison with the non-methylated counterparts. The proteins and DNAs were incubated for 30 min in in suitable buffer and the bound and unbound bands were resolved in 12% polyacrylamide gels. the DNAs were purified from the bound and unbound bands and sequenced.
2017-04-01 | GSE95526 | GEO