Overview of CRISPR/Cas9 systems




CRISPR/Cas9 systems- Background:

The clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR/Cas) systems has been developed as a powerful molecular technology for plentiful areas of biological research 1. The systems are used by various bacteria and archaea to defend against viruses and other foreign nucleic acids and characterized by tremendous diversity in cas genes, which evolved as a response to selective pressure from their viral invaders and other pathogens 2.  This diversity created a broad range of Cas effector proteins, which are significantly varied structurally and functionally. The most interest so far was attracted to Class 2 of the proteins containing a signature Cas9 effector protein, that is in contrast to Class 1 effectors represented by multi-subunit complexes is a single-polypeptide 2, 3, 4, 5. This supported the adaptation of Cas9 which can locate, bind, and cleave double-stranded DNA (dsDNA) targets complementary to its guide crRNA for a wide-range of gene editing applications in mammals shown an incredible impact on many disciplines of human genetics (figure below) .



CRISPR/Cas9 composition

CRISPR consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9) or other orthologue (see below). The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified 2, 3, 4, 5. Thus, one can change the genomic target of Cas9 by simply changing the targeting sequence present in the gRNA. CRISPR/Cas9 system can be employed for gene-knockouts, gene activation, repression, epigenetic gene editing (methylation, demethylation) and single-base nucleotide changes (see figure above) 5, 6, 7, 8, 1, 9. Furthermore, the ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies and has been recently utilized for genome-wide screens 10, 11.

The target specificity between the gRNA and the complementary locus requires a presence of Protospacer Adjacent Motif (PAM) that sited downstream and upstream from the target (for Streptococcus pyogenes Cas9 and Cpf1, respectively) 2, 3, 4, 5, 12. The PAM sequence is necessary for target binding and the exact sequence is dependent upon the species of Cas9 (5′ NGG 3′ for Streptococcus pyogenes Cas9 and NNGRRT for Cpf1) 2, 5, 12. Once expressed, the Cas9 protein and the gRNA form a riboprotein complex. The Cas9-gRNA complex will bind any genomic sequence with a PAM, but the extent to which the gRNA spacer matches the target DNA determines whether Cas9 will cleave DNA. The zipper-like mechanisms of spacer-photospacer binding has been proposed shown the absolute requirement of 10-PAM 5’-adjoined nucleotides called a “seed” sequence 4, 5. Any change in the seed sequences may negatively affect the affinity of DNA-spacer interactions. Furthermore, Cas9 will only cleave the target if sufficient homology exists between the gRNA spacer and target sequences. The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH, which cut the opposite strands of the target DNA 2, 5, 3, 4.

DNA repair pathways activated by CRISPR/Cas9- mediated DSBs

The cleavage will result in a double strand breaks (DSBs) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence) 2, 5, 4. Two DNA repair machineries: error-prone non-homologous end joining (NHEJ) and less efficient, but highly accurate homology directed repair (HDR) are undergo activation following DNA break. The NHEJ repair pathway is the most active repair mechanism, capable of rapidly repairing DSBs. The pathway frequently results in small nucleotide insertions or deletions (InDels) at the DSB site and in most cases, NHEJ gives rise to small InDels in the target DNA which result in in-frame amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene.

By contrast, the DSBs can be repaired by HDR- pathway. To insert or replace a DNA sequence near the break site, a DNA fragment to be used as a template for repair is introduced (reviewed in 1. The repair template contains homology to the regions flanking the DSB- repair of the break leads to insertion of the repair template without introducing extraneous bases. Thus via HDR, scarless insertion of DNA can be introduced to create precise deletions, base substitutions, or insertion of coding sequences for epitope tags, such as fluorescent proteins. This technology has also been applied to engineering genomes of other eukaryotes, such as yeast, flies and zebrafish. The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. To enhance HDR, enabling the insertion of precise genetic modifications, several approaches were utilized 13, 14. The researchers demonstrated that suppression of the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the co-expression of adenovirus 4 E1B55K and E4orf6 proteins might improve the efficiency of HDR 5 to10- fold 14. The low efficiency of HDR has several important practical implications. First, since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally, and if necessary, isolate clones containing the desired genotype. The two pathways of DNA repair highlighted in the figure that below:


CRISPR/Cas9 is highly specific when gRNAs are designed correctly, but specificity is still a major concern, particularly as CRISPR is being developed for clinical use. The specificity of the CRISPR system is determined in large part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and need to be considered when designing a gRNA for your experiment 3, 4.

.In addition to optimizing gRNA design, specificity of the CRISPR system can also be increased through modifications to Cas9 itself. First, Cas9 generates DSBs through the combined activity of two nuclease domains, RuvC and HNH. The exact amino acid residues within each nuclease domain that are critical for endonuclease activity are known (D10A for RuvC and H840A for HNH in S. pyogenes Cas9) and modified versions of the Cas9 enzyme containing only one active catalytic domain (called “Cas9 nickase”) have been generated 3, 4. Cas9 nickases still bind DNA based on gRNA specificity, but nickases are only capable of cutting one of the DNA strands, resulting in a “nick”, or single strand break, instead of a DSB. DNA nicks are rapidly repaired by HDR using the intact complementary DNA strand as the template. Thus, two nickases targeting opposite strands are required to generate a DSB within the target DNA (often referred to as a “double nick” or “dual nickase” CRISPR system) 3, 4, 15. This requirement dramatically increases target specificity. Second, more recently discovered Cpf1 endonuclease demonstrated to be more specific than the SpCas9 enzyme 16, 12. Furthermore, efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of SpCas9-HF1 and eSpCas9 (1.0 and 1.1) variants that contain amino acid substitutions predicted to weaken the energetics of target site recognition and cleavages 17, 18. Using single-molecule Förster resonance energy transfer (smFRET) experiments, Doudna’s group most recently shown that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets 19. They found that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, the researchers designed a new hyper-accurate Cas9 variant (HypaCas9) that has demonstrated high genome-wide specificity without compromising on-target activity in human cells 19.


Activation or Repression of Target Genes Using CRISPR/Cas9

In addition to utility of CRISPR/Cas9 for direct manipulation within DNA sequences, investigators modified the system to alter the regulation of a target gene. One approach to increase expression of a specific gene is to tether the dead- version of Cas9 (harbored D10A and H840A mutations) dCas9:sgRNA complex to a transcriptional activator and program it to bind near the transcriptional start site of a gene of interest 7, 9, 6, 20, 21. For example, the transcriptional activation domain VP64, which consists of four tandem copies of the Herpes Simplex Viral Protein 16 (VP16), attached to the C terminus of dCas9 can be used to increase the expression of a wide variety of different genes. Similarly, KRAB repressor can be fused to the dCas9 creating a robust platform for gene-specific transcriptional silencing. Recently, more sophisticated approaches has been developed for highly efficient regulation of gene-expression programs. These include- coexpression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, dCas9 fused to several different activation domains in series (e.g. dCas9-VPR) or co-expression of dCas9-VP64 with a “modified scaffold” gRNA and additional RNA-binding “helper activators” (e.g. SAM activators) 22, 23, 9, 6.  Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.



Activation and repression of a target gene: RNA-programmed gene activators can be assembled through the direct fusion of dCas9 with the transcriptional activators VP64, or indirect- via MS2 binding motif- with transcriptional activators p65-HSF, NF-kb and others (not shown here- the histone acetyltransferase enzyme p300 or the DNA demethylase Tet1) 7, 9, 6, 20, 21..  Alternately, transcriptional repressors such as KRAB, de-novo methyltransferase 3A and the histone demethylase LSD1 can be fused either directly or indirectly to repress a target gene 20, 24, 25.

Delivery platforms

Many viral and non-viral methods for delivery of CRISPR/Cas9 gene-editing tools has been recently developed (reviewed in 26, 27, 28). The obvious advantage of viral-mediated gene-transfer is that it deliver a therapeutic cargo in highly efficient manner, currently unachievable by non-viral methods and strategies. Furthermore, initially safety concerns including high immunogenicity and an increased risk of insertional mutagenesis have been recently addressed with the development of more advance and safe viral tools 29, 30.

Non-viral vectors are generally lower in efficiency but have the advantage of diverse available chemistry, capacity for functionalization and targeting, and ease of manufacturing. Both delivery systems have seen success in specific applications. The main advantage of non-viral mediated gene-delivery stems from its transient capability. Indeed, transient platforms including DNA plasmids, messenger RNA (mRNA), and ribonucleoprotein (RNP)-molecules utilized for CRISPR/Cas9 delivery have been shown to be highly advantageous for minimizing non-specific effects of the CRISPR/Cas9 system (reviewed in 26, 27). Indeed, the high turnover rate of rapidly degraded transiently delivered CRISPR/Cas9 corresponded to a reduced rate of off-target mutations. Furthermore, Cas9-sgRNA RNPs-mediated DNA cleavage is followed by their almost instant degradation and clearance from the cells, which suggests that gene editing might only require a short-term presence of its components 31.


However, low transduction efficiency remains a serious limitation of RNPs and other non-viral delivery platforms. To overcome this limitation, a lentivirus-pre-packaged Cas9 protein (Cas9P LV) system was developed recently and was shown to be effective for disruption of gene expression in naïve T cells32. Significantly, transiently delivered Cas9 showed high target specificity and induced no measurable InDels at off-target DNA sites. However, production titers of the prepackaged-Cas9 system were observed to be lower than those of conventional LVs32.


Lentiviral vectors (LVs) are an important means of delivering CRISPR/Cas9 components due to their ability to accommodate large DNA payloads and efficiently transduce a wide range of dividing and non-dividing cells. LVs also display low cytotoxicity and immunogenicity and have a minimal impact on the life cycle of the transduced cells (reviewed in 29). Such features have led to LVs being used as the gene-editing regimen of choice to treat HIV-1, HBV and HSV-1 infections, as well as to correct defects underlying human hereditary diseases, such as cystic fibrosis 33, 34, 35, 36. Despite these successes, this system suffers from significant drawbacks. Permanently expressed CRISPR/Cas9 may facilitate undesirable off-target effects, hindering their utility for genome-editing applications that require high levels of precision. Indeed, rise of promiscuous interactions with off-target genes due to excess gRNA/Cas9 is well-documented 10, 31. Furthermore, sustained expression of gRNA/Cas9 in vitro increases the tolerability of mismatches in the guide-matching region and the protospacer adjacent motif (PAM), thereby promoting non-specific double-strand breaks (DSBs) 37, 38. Along the same lines, the ratio of insertions and deletions (InDels) at off-target vs. target sites in vivo increases with higher Cas9 and gRNA concentrations 3.  These observations suggest that non-integrating vectors would be desired for delivery CRISPR/Cas9 components if the enhance safety of gene-editing manipulations are pursued.


Integrase-deficient lentiviral vectors

Integrase-deficient lentiviral vectors (IDLVs) have garnered significant interest among researchers for precise in vivo analysis of genetic diseases, since they significantly reduce the risk of insertional mutagenesis inherent in integrating delivery platforms (reviewed in {Kantor, 2014 #53}, {Kantor, 2011 #26}, {Nelson, 2016 #66}. IDLVs are an ideal platform for delivery of large genetic cargos where only transient expression of the transgene is desired (reviewed in 6,  22, 23, 24),  25, 26, 27, 28, 29, 30, 31, 32.  IDLVs were successfully employed in the past in mouse models as gene replacement therapies for degenerative retinal disease, hemophilia B (36, 37); they show high efficacy in cancer immunotherapy- setting as a means of inducing protective immune responses to human pathogens 38, 39, 40. Furthermore, a growing body of literature describes IDLVs carrying zinc-finger nucleases as an effective means of gene editing for clinical and basic science applications 28, 30, 31, 32. For instance, Lombardo and colleagues have successfully employed non-integrating vectors as a means of avoiding genotoxicity associated with continuous expression of zinc-finger nucleases (ZFNs), and for delivering the donor DNA template required for DNA repair-mediated gene editing 28. These researchers demonstrated that the IDLV-ZFNs system is capable of effectively disrupting expression from the gene encoding the HIV-1-coreceptor CCR5. Additionally, Joglekar and colleagues30 successfully employed IDLVs to deliver ZFNs and donor templates for site-specific gene modification at the human adenosine deaminase (hADA) locus in primary T-lymphocytes. Most recently, Hoban and colleagues demonstrated efficient gene editing of the mutated human β-globin gene in CD34+ hematopoietic stem and progenitor cells by co-delivering CRISPR/Cas9 reagents and donor templates via IDLVs41.

We recently developed novel IDLVs for delivering Cas9 and sgRNA through a single vector and demonstrated that the novel vector enables facile and robust gene editing in in-vitro and in-vivo settings – which is particularly advantageous for developing a translatable gene therapy products {Ortinski, 2017 #83}. Furthermore, we demonstrate that the IDLV-CRISPR/Cas9 system is expressed transiently and has a significantly lower capacity to induce off-target mutations than its integrating counterparts have {Ortinski, 2017 #83}.


The image copied from Ortinski et al. 2017; published in Molecular Therapy Methods and Clinical Development.  (A) A schematic map of the vector cassette. A shorter version of pLentiCRISPRv2 was created to include a unique BsrGI restriction enzyme site flanked by two BsmBI sites to be used for cloning sgRNAs. Other regulatory elements of the vectors include a primer-binding site (PBS), splice donor (SD) and splice acceptor (SA), central polypurine tract (cPPT) and PPT, Rev Response element (RRE), WPRE, and the retroviral vector-packaging element, psi (ψ) signal. A human cytomegalovirus (hCMV) promoter, a core-elongation factor 1α promoter (EFS-NC), and a human U6 promoter are highlighted. (B) Production titers of the vectors with (novel) and without (parental) Sp1 binding sites as determined by p24ELISA assay. The results shown increase in production of novel IDLVs and (C) ICLV vectors (the functional titer has been determined by counting puromycin-resistant colonies).


Adenoviruses and adeno-associated vectors (AAVs)

Adenoviruses and AAVs have also been broadly investigated for their potential use in vitro and ex vivo (reviewed in {Kantor, 2014 #53}, {Kantor, 2011 #26}). The production titers are traditionally high with AAVs which makes this viral platform to be the most used viral vector for genome engineering to date (reviewed in 26). In addition, being transient delivery platform highlights one of the main advantages of AAV vectors. First publications utilized AAV for CRISPR/Cas9 delivery used SpCas9- for example,  Platt and colleagues39 successfully packaged the endonuclease and sgRNA into viral particles for in vivo modeling of loss-of-function mutations in P53 and LKB1 genes in mouse lung adenocarcinomas. However, the large size of the SpCas9 gene (4.2kb) imposes a significant burden on the packaging capacity of AAVs. To overcome this bottleneck, Gang Bao’s group recently developed a split-intein Cas9 system that can be separated into two AAV cassettes 40. This approach allows for increased in overall packaging capacity but necessitates production and co-transduction of two AAV vectors. The discovery of a shorter, but equally potent Cas9 enzyme derived from Staphylococcus aureus (SaCas9) (see above) led to development of SaCas9/guide RNA system that could be efficiently packaged and delivered by AAV vectors 41. This system was shown to efficiently target the cholesterol regulatory gene PCSK9 in the mouse liver 41.


The Duke Viral Vector Core is pleased to offer a comprehensive package of services involved creation and production of customized AAV, IDLV and LV/RV vectors for delivery CRISPR/Cas9 gene-editing tools. Our CRISPR/Cas9 vectors are suitable for both in-vitro and in-vivo studies. In addition, we guarantee highest- quality standards and safety of the vectors. The core owns comprehensive collection of CRISPR/Cas9 plasmids acquired from Addgene and created at the facility. Our vectors are production/titer optimized.


CRISPR/Cas9 plasmids received from Addgene are not available for the distribution.


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Note: CRISPR/Cas9 plasmids received from Addgene are not available for the distribution

The core has a comprehensive collection of LVs and AAVs vectors expressed CRISPR-Cas9 components for gene editing- gRNA with or without Cas9 nuclease; WT-Cas9; nickase-Cas9; inducible-Cas9 and dCas9.

The collection derives from in-house developed expression cassettes (backbone-optimized & titer-improved) (please see Ortinski et al, 2017 in Molecular Therapy Methods and Clinical Dev) http://www.cell.com/molecular-therapy-family/methods/abstract/S2329-0501(17)30057-8 or obtained from other investigators via Addgene repository. The vectors contain fluorescent markers for direct visualization of expression and/or antibiotics-resistance genes (puromicin, blastidin and others) which can be used if selection of transduced cells is desired. The vectors support single or paired gRNA-CRISPR approach for efficient gene editing manipulations. Most of the viral platforms are validated- we will be able to guarantee high-level of CRISPR/Cas9 expression.