Cas9 Binding Research Articles

Multiplexed single-molecule experiments reveal Cas9 nucleosome invasion dynamics

For genome editing applications in eukaryotic cells, the Cas9 nuclease must target chromatinized DNA. The position of PAM sites within nucleosomes, the presence of histone modifications as well as sgRNA specificity are important factors for Cas9 recognition and binding, but the mechanisms governing Cas9/dCas9 chromatin invasion remain unclear. Single-molecule measurements provide detailed mechanistic insight into intricate molecular processes, and are thus suitable to reveal how Cas9 dynamically engages nucleosomes.

Bovine Genome Analysis to Unravel the Location and Feature of Target Sites of RNA-Guided Hyperactivated Recombinase Gin with Spacer Length Six

Background: Programmable nucleases are very promising tools of genome editing (GE), but they suffer from limitations including potential risk of genotoxicity which led to the exploration of safer approach of GE based on RNA-guided recombinase (RGR) platform. RNA-guided recombinase (RGR) platform operates on a typical recognition or target site comprised of the minimal pseudo-core recombinase site, a 5 to 6-base pair spacer flanking it and whole this central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites followed by protospacer adjacent motifs (PAMs). Methods: The current study focuses on analysis of entire cattle genome to prepare a detailed map of target sites for RNA-guided hyperactivated recombinase Gin with spacer length six. For this, chromosome wise whole genomic sequence data was retrieved from Ensembl. After that search pattern for recombinase Gin with spacer length six was designed. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. Result: Total number of RGR target sites identified in bovine genome for recombinase Gin was 677 with spacer length six. It was also investigated that whether these RGR target sites are present with in any gene or not and it was found that RGR target sites lies in both genic and intergenic region. Besides this, description of genes in context with these target sites was identified.

DCas9 binding inhibits the initiation of base excision repair in vitro

Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K D ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.

CRISPR/Cas9-Mediated Epigenome Editing of the IL-10 Gene for Targeted Whole Organ Gene Therapy for Lung Transplant

<h3>Purpose</h3> Anti-inflammatory and immunomodulatory gene therapy has shown promise in lung transplant. CRISPR/Cas9 technology unleashed the possibility of making gene therapy persistent by radically altering gene expression through precise DNA targeting. We envisioned activation of the endogenous IL-10 gene, which codes for this anti-inflammatory cytokine, using epigenome editing via CRISPR/Cas9 in the lung. This study investigated the efficacy and specificity in vitro and in rat lungs. <h3>Methods</h3> Adenoviral vectors were developed expressing a Cas9 activator and either one or two gRNAs targeting the promoter region of the IL-10 gene in the genome (Fig. A). Lewis rats were divided into three groups (Fig. B, n=5-10), trans-bronchially administered adenovirus (1 × 10⁸ PFU/rat) under standard transplant triple immunosuppression, and assessed after 72 hours. For specificity, primary lung fibroblasts treated with adenovirus were analyzed at 48 hours using chromatin immunoprecipitation (ChIP) of Cas9 followed by qPCR designed against the on- and off-target regions. <h3>Results</h3> The two-gRNA group showed significantly increased IL-10 gene expression compared to the diluent group (relative expression: 19.0±10.8 vs 1.3±1.2, p=0.001) and the single gRNA group (2.9±0.78, p=0.0009) (Fig. C). There was no significant increase in TNFα or IL-6 gene expression or Wet/Dry ratio in the two-gRNA group compared to the diluent group (Fig. D, E) suggesting minimal vector-related inflammation. ChIP-qPCR demonstrated enriched binding of Cas9 to on-target sites (Fig. F, fold enrichment; 17.5±1.7, p=0.02, 18.5±5.8, p=0.02, respectively) compared to the control region whereas the binding to off-target sites were not significantly enriched. <h3>Conclusion</h3> We have achieved targeted in vivo whole organ activation of the IL-10 gene via CRISPR/Cas9-mediated epigenome editing in the lung. This study demonstrates the feasibility of this strategy for optimizing and immunologically enhancing donor organs for transplant.

Genome oligopaint via local denaturation fluorescence in situ hybridization

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement.

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to "proofreading" capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR.

CRISPR-Cas9 has transformed biomedical research and medicine through convenient and targeted manipulation of DNA. Time- and spatially-resolved control over Cas9 activity through the recently developed very fast CRISPR (vfCRISPR) system have facilitated comprehensive studies of DNA damage and repair. Understanding the fundamental principles of Cas9 binding and cleavage behavior is essential before the widespread use of these systems and can be readily accomplished in vitro through both cleavage and electrophoretic mobility shift assays (EMSA). The protocol for in vitro cleavage consists of Cas9 with guide RNA (gRNA) ribonucleoprotein (RNP) formation, followed by incubation with target DNA. For EMSA, this reaction is directly loaded onto an agarose gel for visualization of the target DNA band that is shifted due to binding by the Cas9 RNP. To assay for cleavage, Proteinase K is added to degrade the RNP, allowing target DNA (cleaved and/or uncleaved) to migrate consistently with its molecular weight. Heating at 95°C rapidly inactivates the RNP on demand, allowing time-resolved measurements of Cas9 cleavage kinetics. This protocol facilitates the characterization of the light-activation mechanism of photocaged vfCRISPR gRNA.

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein.

Optogenetic control of CRISPR–Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.

CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.

While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53−/− cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.

Appropriate Delivery of the CRISPR/Cas9 System through the Nonlysosomal Route: Application for Therapeutic GeneEditing.

The development of gene delivery has attracted increasing attention, especially when the introduction and application of the CRISPR/Cas9 gene editing system appears promising for gene therapy. However, ensuring biosafety and high gene editing efficiency at the same time poses a great challenge for its in vivo applications. Herein, a pardaxin peptide (PAR)‐modified cationic liposome (PAR‐Lipo) is developed. The results are indicative that significantly enhanced gene editing efficiency can be obtained through the mediation of PAR‐Lipos compared to non‐Lipos (non‐PAR‐modified liposomes) and Lipofectamine 2000, owing to its protection toward carried nucleotide by the prevention of lysosomal capture, prolongation of retention time in cells through the accumulation in the endoplasmic reticulum (ER), and more importantly, facilitation of the nuclear access via an ER‐nucleus route. Accumulation of PAR‐Lipos in the ER may improve the binding of Cas9 and sgRNA, thus further contributing to the eventually enhanced gene editing efficiency. Given their high biosafety, PAR‐Lipos are used to mediate the knockout of the oncogene CDC6 in vivo, which results in significant tumor growth inhibition. This work may provide a useful reference for enhancing the delivery of gene editing systems, thus improving the potential for their future clinical applications.

Core Hairpin Structure of SpCas9 sgRNA Functions in a Sequence- and Spatial Conformation-Dependent Manner.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a widely used genome-editing tool with great clinical potential. However, its application is limited because of low editing efficiency of some target sequences and off-target effects. As this system contains only the Cas9 protein and a single-guide RNA (sgRNA; engineered from crRNA and tracrRNA), the structure and function of these components should be studied in detail to address the current clinical needs. Consequently, we investigated the structural and sequence features of the core hairpin (the first stem loop of sgRNA) of SpCas9 sgRNA. We showed that the core hairpin structure of sgRNA is essential for SpCas9/sgRNA-mediated DNA cleavage and that the internal loop structure in the core hairpin plays a vital role in target DNA cleavage. We observed that the root stem structure within the core hairpin preferentially forms Watson-Crick base pairs and should be of a specific length to maintain an appropriate spatial conformation for Cas9 binding. However, the length of the leaf stem structure of the core hairpin is flexible, having a variable nucleotide composition. Furthermore, extension of the leaf stem structure enhances the DNA cleavage activity of the Cas9/sgRNA complex, and this could be used to enhance the efficiency of gene editing. These observations provide insight into the sgRNA/Cas9 interaction, indicating that sgRNA modification could be a strategy for improved DNA editing efficiency, and optimized sgRNA can be further used for genome-wide functional screening and clinical application.

Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.

In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing.

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

Discovery and Evaluation of Peptide Ligands for Selective Adsorption and Release of Cas9 Nuclease on Solid Substrates.

The rapid expansion of CRISPR in biotechnology, medicine, and bioprocessing poses an urgent need for advanced manufacturing of Cas nucleases. The lack of Cas-targeting ligands, however, prevents the development of platform processes for purifying this class of molecules. This work represents the first effort at developing short synthetic Cas9-binding peptides and demonstrates their applicability as affinity ligands for the purification of a Cas nuclease. Candidate Cas9-targeting peptides were initially identified by screening a solid-phase peptide library against a model mixture of Streptococcus pyogenes Cas9 spiked in Escherichia coli cell lysate. An ensemble of homologous sequences was identified, conjugated on Toyopearl resin, and evaluated by Cas9 binding studies to identify sequences providing selective Cas9 capture and efficient release. In silico docking studies were also performed to evaluate the binding energy and site of the various peptides on Cas9. Notably, sequences GYYRYSEY and YYHRHGLQ were shown to target the RecII domain of Cas9, which is not involved in nuclease activity and was targeted as an ideal binding site. The peptide ligands were validated by purifying Cas9 from the E. coli lysate in dynamic conditions and through measurements of binding capacity and strength (Qmax and KD). The resulting values of Qmax = 4-5 mg Cas9 per mL of resin and KD ∼ 0.1-0.3 μM, product recovery (86-89%), and purity (91%-93%) indicate that both peptides, and YYHRHGLQ in particular, can serve as capture ligands in a platform purification process of Cas9.

AcrIIA5 Inhibits a Broad Range of Cas9 Orthologs by Preventing DNA Target Cleavage

CRISPR-Cas9 is an adaptive immune system for prokaryotes to defend against invasive genetic elements such as phages and has been used as a powerful tool for genome editing and modulation. To overcome CRISPR immunity, phages encode anti-CRISPR proteins (Acrs) to inhibit Cas9, providing an efficient "off-switch" tool for Cas9-based applications. Here, we characterized AcrIIA5, which is a Cas9 inhibitor discovered in a virulent phage of Streptococcus thermophilus. We found that AcrIIA5 is a potent and broad-spectrum inhibitor of CRISPR-Cas9, which can inhibit diverse Cas9 orthologs of type II-A, type II-B, and type II-C. AcrIIA5 inhibits Cas9 by preventing DNA target cleavage, but DNA target binding of Cas9 is unaffected. Importantly, it can affect the activity of the RuvC nuclease domain of Cas9 independent of the HNH nuclease domain. Our work expands the diversity of the inhibitory mechanisms used by Acrs and provides the guidance for developing controlling tools in Cas9-based applications.

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

Exploration of RNA-Guided Recombinase Target Sites for Hyperactivated Recombinase Beta in Bovine Genome

The present study was conducted to determine the effect of different cooling systems; Fan Fogger (FF) and Fan Pad (FP) on micro environment of poultry house, thermal comfort, welfare, egg production and egg quality parameters of laying hens. This experiment was conducted on 210, White Leghorn laying pullets (32 weeks old) during hot-dry summer months (May - July) under deep litter system of housing. The FP and FF cooling systems significantly dropped the mean shed temperature and increased the relative humidity. Thus, better THI resulted in increase in egg production by 4.66 % and 3.32 % under FP and FF systems over the control group. However, specific gravity, H.U, egg shell thickness, yolk index and yolk color were not significantly influenced by cooling treatments. Significantly lower levels of antioxidant enzymes viz. LPO, Catalase, G6PD, GPx and SOD was registered in cooling groups. Both the cooling devices contributed towards bird welfare by altering the behavioral expression from agonistic to non-agonistic activities.RNA-guided recombinases (RGR) are potentially valuable tools for basic research and genetic modifications. The platform has been demonstrated to do genome editing efficiently. The platform operates on a typical recognition site comprised of the degenerate recombinase site, a 5 to 6-base pair spacer flanking it and this whole central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites which is followed by protospacer adjacent motifs. In present investigation, a detailed map of target sites for RNA-guided recombinase platforms based on hyperactivated recombinase Beta throughout the bovine genome was prepared. For this, Chromosome wise whole genomic sequence data was retrieved from Ensembl followed by designing search pattern for recombinase Beta with spacer length five. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. In total,436 RGR target sites were identified in bovine genome for recombinase Beta with spacer length five. These RGR target site provide potential of being utilized for specific genomic integration, deletion or inversion.