CRISPR and RNAi
Developing therapeutics for genetic disorders heavily relies on correlating genetic expression to phenotype. So far, the best way to achieve this has been to block the expression of a gene and analyze its effect on phenotype. To experimentally regulate gene expression and interrogate gene function, two methods, RNAi and CRISPR, have emerged as the most popular methods in the past few years.
CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.
The main distinction between RNAi and CRISPR-Cas9 is that RNAi reduces or knockdowns gene expression at the post-transcriptional level by targeting RNA, whereas CRISPR-Cas9 is a gene-editing tool that targets DNA to permanently alter or knockout gene expression. Knockdown with RNAi produces a hypomorphic phenotype in contrast to the true null knockout with CRISPR-Cas9. Both options can be useful depending on the experimental design and question to be addressed. A complete knockout with CRISPR-Cas9 may in some cases be required, preventing any uncertain effects of residual low-level protein expression that remains after knockdown, which may mask certain phenotypes. It could prove useful in cell lines with polyploidy, such as cancer cell lines with multiple gene copies. However, in some cases knockdown could be advantageous. For example, knockout of essential genes is cell lethal, making effects on these difficult to study. Knockdown rather than knockout can also better mimic the inhibition of a target by a drug, allowing reduction of protein levels and intricacies of gene expression on phenotype to be studied.
But the development and expansion of the CRISPR-Cas9 toolkit means that the choice between knockout and knockdown is now not just between CRISPR-Cas9 and RNAi. The use of a catalytically inactive version of Cas9 (dCas9) tethered to a transcriptional repressor has provided the means to perform CRISPR-mediated knockdown. CRISPR interference (CRISPRi) combines the knockdown capacity of RNAi with the efficiency and specificity of CRISPR for functional genomic screening. Recent computational efforts to identify new CRISPR systems uncovered a novel type of RNA targeting enzyme, Cas13. The diverse Cas13 family contains at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d.
Fit CRISPR into AAV vectors
In general, delivering over-sized transgenes has been a longstanding hurdle in the AAV gene therapy field. AAV can only package ~5.0 kb of genome. The widely used, SpCas9 is 4.2 kb and therefore necessitates the use of short gene regulatory elements including promoter and polyadenylation signal totaling less than 0.5 kb. This excludes some promoters that have desired expression strength and specificity. Furthermore, other genome editing components must be carried on a separate AAV vector, such as sgRNA expression cassette(s) and donor template for HDR or gene insertion. A dual-vector delivery scheme can achieve genome editing only when both vectors are taken up by the same cell, potentially limiting editing efficiency. An early approach to overcome this challenge was to identify smaller orthologs of Cas9. Additional smaller Cas proteins have since been discovered or engineered and experimentally validated as effective genome editing tools in mammalian cells. These Cas proteins are more compatible with AAV delivery, enabling additional vector design options like expanded promoter choices and a streamlined delivery scheme. For example, SaCas9 has a gene size of 3.2 kb, allowing a single AAV vector to express SaCas9 together with one or two sgRNAs.
DNA Editing by CRISPR
CRISPR-Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, and tracrRNA along with an optional section of DNA repair template that is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).
Cas9: An enzyme whose active form is able to modify DNA. Many variants exist with different functions (i.e. single-strand nicking, double-strand breaking, DNA binding) due to each enzyme's DNA site recognition function.
Repair template: DNA molecule used as a template in the host cell's DNA repair process, allowing insertion of a specific DNA sequence into the host segment broken by Cas9.
CRISPR-Cas9 often employs a plasmid or a virus to transfect or infect the target cells. The main components of this plasmid/virus are displayed in the image and listed in the table. The crRNA is uniquely designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to specific sequences within the host cell's DNA. The crRNA must bind only where editing is desired. The repair template is also uniquely designed for each application, as it must complement to some degree the DNA sequences on either side of the cut and also contain whatever sequence is desired for insertion into the host genome.
Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).This sgRNA can be included alongside the gene that codes for the Cas9 protein and made into a plasmid in order to be transfected into cells. Many online tools are available to aid in designing effective sgRNA sequences
RNA Editing by Cas13
DNA targeting CRISPR enzymes, such as Cas9 and Cas12a (formerly Cpf1), have enabled many new possibilities for manipulating and studying DNA. Recent computational efforts to identify new CRISPR systems uncovered a novel type of RNA targeting enzyme, Cas13. The diverse Cas13 family contains at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d.
Cas13s function similarly to Cas9, using a ~64-nt guide RNA to encode target specificity. The Cas13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by a 28 – 30-nt spacer that is complementary to the target region. In addition to programmable RNase activity, all Cas13s exhibit collateral activity after recognition and cleavage of a target transcript, leading to non-specific degradation of any nearby transcripts regardless of complementarity to the spacer.
Many applications have also been built using Cas13s in mammalian cells, including transcript knockdown, live-cell transcript imaging, and RNA base editing.
While Cas13a showed some activity for RNA knockdown, certain orthologs of Cas13b proved more stable and robust in mammalian cells for RNA knockdown and editing. More recently, additional orthologs of Cas13 have been discovered, including Cas13d, which has been leveraged for efficient and robust knockdown across many endogenous transcripts. Cas13d can be used to modulate splicing of endogenous transcripts and that the coding sequence for Cas13d is small enough to fit within the packaging limits of AAV for in vivo delivery.
Beyond these in vivo activities, Cas13s non-specific RNase activity, could be leveraged to cleave fluorescent reporters upon target recognition, allowing for the design of sensitive and specific diagnostics using Cas13.
One of the most straightforward applications of Cas13 in vivo is targeted RNA knockdown using mammalian codon optimized Cas13 and guide expression vectors. Knockdown of RNA relies on cleavage of the targeted transcripts by the endogenous RNase activity of the dual HEPN domains of the protein, the efficiency of which varies between different orthologs and subtypes of Cas13. As a result, guide design and restrictions on targeting depend on the system used.
Beyond the above design guidelines, the many different Cas13 subtypes have varied activities in different model systems. Because of the variety of Cas13 subtypes and orthologs, selection of the right construct for knockdown can be difficult. While LwaCas13a, PspCas13, and RfxCas13d have all been demonstrated to achieve robust knockdown across numerous genes in mammalian cells, a comparison between Cas13a, Cas13b, and Cas13d indicated that RfxCas13d has the most robust and substantial knockdown in HEK293T cells. For plant applications, only LwaCas13a has been tested, where it achieved substantial knockdown in rice protoplasts.
sgRNA selection services
At AAVnerGene, we will help customer select sgRNAs for different Cas proteins. In each selection service, 5 sgRNA plamsids, one control sgRNA plasmid and one transgene-reporter fusion plasmid are generated. Then, the sgRNA plamsid, the transgene-reporter fusion plasmid and the corresponding Cas plasmid are cotransfected into targeting cells. The gene knockdown efficiency is measured by the reporter gene expression from the target-reporter fusion plamsid. The 2nd reporter gene in the sgRNA plamsid is used for internal control.
Customer can select the Cas genes(SpCas9, SaCa9, LwaCas13a, PspCas13, RfxCas13d, etc.) promoters(CMV, CAG, TTR, hSyn1, etc.), reporters(EGFP, mCherry, Gluc, Cluc, Rluc, Fluc, etc.), and cell lines(Hela, HEK 293, Huh7, etc.), accordingly.
Also, customers can select the sgRNA candidates by themselves.
Please contact us to get a quote.
shRNA selection services
At AAVnerGene, we provide shRNA selection services. In each package, 5 shRNA plamsids, one control shRNA plasmid and one transgene-reporter fusion plasmid are included. The transgene-reporter fusion plasmid and shRNA plasmid are cotransfected into targeting cells. The gene knockdown efficiency is measured by the reporter gene expression from the target-reporter fusion plamsid. The 2nd reporter gene in the shRNA plamsid is used for internal control.
Customer can select the promoters(CMV, CAG, TTR, hSyn1, etc.), reporters(EGFP, mCherry, Gluc, Cluc, Rluc, Fluc, etc.), and cell lines(Hela, HEK 293, Huh7, etc.), accordingly. Please contact us to get a quote.
Also, customers can select the sgRNA candidates by themselves.
Please contact us to get a quote.