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CRISPR/Cas9 Methods

What does CRISPR stand for?

RNA-guided engineered nucleases (RGENs; the most widely used is the Cas9 protein from Streptococcus pyogenes) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeats (also known as RSRS (Francisco Mojica in 1993)) have revolutionized the field of gen manipulation by offering a powerful method for disrupting genes of interest. The technique enables genome editing in human cell lines, animals, and plants, but show limitations by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. 

To overcome the problem with these negative on- and off-target effects, it is possible to deliver purified recombinant Cas9 protein together with guide RNA into cultured human cells by co-transfection.

Publications have shown that it is possible to induce site-specific mutations at frequencies up to 80%, while reducing off-target mutations associated with plasmid transfection. RGEN ribonucleoproteins cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection. CRISPR Cas9

The CRISPR system consists of the guide RNA (gRNA/sgRNA) and a non-specific CRISPR-associated endonuclease (e.g. Cas9 protein from Streptococcus pyogenes that is currently the most widely used in genome engineering). sgRNA is a short synthetic RNA composed of a sequence (tracrRNA = non-target specific sequence) necessary to bind to the Cas9 protein and a gene specific ca. 20 nucleotide “spacer” or “targeting” sequence (crRNA) which defines the genomic target to be modified. CRISPR spacers recognize and cut exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms.

Although other programmable editing tools, such as zinc finger nucleases and transcription activator-like effector nucleases, have significantly improved the capacity for precise genome modification, these techniques have limitations and are especially very time consuming. CRISPR/Cas9 technology represents a significant improvement over these other next-generation genome editing tools, offering an unmet level of targeting, efficiency, and ease of use.

Able to achieve highly flexible and specific targeting, CRISPR-Cas systems can be modified and redirected to become a powerful tool for genome editing in virtually any organism, including mammalian cells. Actually Genaxxon offers different products for the CRISPR technology as an optimized pDNA Transfection reagent, and the Cas9 protein.

With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated to become a powerful tool for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it, for example with our GenaxxoFect transfection reagent along with the Cas endonuclease to your target cell.

The CRISPR/Cas system is a prokaryotic adaptive immune response system that uses non-coding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. This DNA damage is repaired by cellular DNA repair mechanisms, either via the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway. Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break.

Knock-out constructs using CRISPR/Cas9

CRISPR/Cas9 can be used to generate knock-out cells. The genomic target can be any ca. 20 nucleotide DNA sequence, provided the sequence is unique compared to the rest of the genome and the target is present immediately upstream of a Protospacer Adjacent Motif (PAM). This PAM sequence is absolutely necessary for target binding. It is 5′ NGG 3′ for Streptococcus pyogenes Cas9. Once expressed, the Cas9 protein and the gRNA form a riboprotein complex which leads to a conformational change of Cas9 that shifts the molecule from an inactive, non-DNA binding conformation, into an active DNA-binding conformation. While binding to Cas9, the “spacer” sequence of the gRNA remains free to interact with target DNA.

Cas9 will only cleave the target if sufficient homology exists between the gRNA spacer and target sequences. The “zipper-like” annealing mechanics may explain why mismatches between the target sequence in the 3′ seed sequence completely abolish target cleavage, whereas mismatches toward the 5′ end are permissive for target cleavage. Upon target binding Cas9 undergoes a second conformational change that positions the nuclease domains to cleave opposite strands of the target DNA resulting in a double strand break (DSB) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by either the efficient but error-prone Non-Homologous End Joining (NHEJ) pathway or the less efficient but high-fidelity Homology Directed Repair (HDR) pathway.

The NHEJ repair pathway is fast but error prone frequently resulting in small nucleotide insertions or deletions (InDels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications as it will result in a diverse array of mutations. 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. Ideally, the end result is a total loss of the function of a target gene, however, the knock-out phenotype for a given mutant cell is ultimately determined by the amount of residual gene function.

Multiplex Genome Engineering with CRISPR/Cas9

Placing several gRNAs on the same plasmid ensures that every cell that takes up a plasmid will also express all of the gRNAs thus increasing the likelihood that all desired genomic edits will be carried out by Cas9. Such “multiplex” CRISPR applications include:

  • The use of dual nickases to generate a knock-out or edit a gene
  • Using Cas9 to generate large genomic deletions
  • Modifying multiple different genes at once

Currently researcher have been successful to target 2 to 7 genetic loci by multiplex CRISPR systems.

For analysing the result the Genaxxon Multiplex PCR Mastermix can be used.

 

References:

Lander ES (Jan 2016). "The Heroes of CRISPR". Cell 164 (1-2): 18–28. doi:10.1016/j.cell.2015.12.041. PMID 26771483.

Mojica FJ, Díez-Villaseñor C, Soria E, Juez G (Apr 2000). "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria". Molecular Microbiology 36 (1): 244–6. doi:10.1046/j.1365-2958.2000.01838.x. PMID 10760181.open access publication.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.

Sojung Kim, Daesik Kim, Seung Woo Cho, Jungeun Kim and Jin-Soo Kim. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Published in Advance April 2, 2014, doi: 10.1101/gr.171322.113. Genome Res. 2014. 24: 1012-1019.

Kent G. Golic. RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model and Nonmodel Organisms. Genetics October 2, 2013 vol. 195 no. 2 303-308; DOI: 10.1534/genetics.113.155093

 
 
 

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