CRISPR/Cas Nuclease RNA-guided Genome Editing

Sigma Aldrich is proud to offer its newest line of genome editing tools, Sigma CRISPRs, to the global research community. As the first company to commercially offer targeted genome editing technology nearly ten years ago, no one has more expertise in this field than Sigma Aldrich. This experience is especially important when it comes to crafting genome editing tools that possess the critical requirements of having specific targeting and robust cutting activity. We provide both in our new Sigma CRISPR product line, and now we put our skill into your hands with a quick and simple web-based design platform. Sigma CRISPR products can be ordered directly through the link below or browse the CRISPR content on this page to learn more about the technology.

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What is CRISPR/Cas?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The discovery of the type II prokaryotic CRISPR “immune system” has allowed for the development for an RNA-guided genome editing tool that is simple, easy and quick to implement.

How does CRISPR/Cas work?

The CRISPR pathway was discovered in bacteria, where it functions much like an immune system against invading viruses and plasmid DNA. Short DNA sequences (spacers) from invading viruses are incorporated at CRISPR loci within the bacterial genome and serve as “memory” of previous infection. Re-infection triggers the complementary mature CRISPR RNA (crRNA) to find a matching sequence – which provides the CRISPR-associated (Cas) nuclease the specificity to form a double-strand break at specific “foreign” DNA sequences.

Genomic Target Site

The Cas9 nuclease and requirements for RNA-guided genome-editing

Cas9 is the nuclease guided by the crRNA and tracrRNA (or trans-activating crRNA) to cleave specific DNA sequences (Deltcheva et al. 2011). A guide RNA (gRNA) can be designed to include a hairpin that mimics the tracrRNA-crRNA complex (Jinek et al. 2012). Binding specificity is based on the gRNA and a three nucleotide NGG sequence called the protospacer adjacent motif (PAM) sequence (Marraffini and Sontheimer, 2010). For site-specific genome editing, the CRISPR/Cas9 system minimally requires the Cas 9 nuclease and the gRNA.

What are the benefits of using CRISPR/Cas9 for genome-editing?

The system is simple, as it only requires a Cas nuclease and a gRNA against the target sequence to function as a site-specific nuclease. Also, despite the bacterial evolutionary origins of the system, data demonstrates high levels of cutting activity in mammalian cells (Cong et al., 2013; Mali et al., 2013), particularly at numerous simultaneous targets, (Wang et al., 2013). In addition, the requirement for an NGG sequence makes target design simple and straightforward in genomic regions where off-targeting is not an issue. Finally, CRISPR provides researchers a fast and cost-effective genome editing tool to use for modifying the genomes of various organisms.

All-in-one, ready-to-use Cas9 and guide RNA (gRNA) expression plasmids

  • A codon optimized Cas9 protein and a gRNA are expressed from a single vector and provided as ready-
    to-use, transfection-grade DNA.
  • Unique CRISPR sites pre-designed to minimize off-targeting are available for the coding regions of the human, mouse, and rat genomes. Custom designs for any other regions or species are always available upon request (see Custom CRISPR request form).
  • GFP or RFP is fused to the C-terminus of Cas9 with a 2A peptide, enabling tracking of transfection efficiency and enrichment of genome editing activity through FAC sorting.
CRISPR/Cas Single Plasmid Format


CRISPR paired nickases

  • Sigma′s CRISPR paired nickases further minimize off-target double-stranded breaks by requiring the independent binding of two separate gRNAs to a localized genomic region.
  • In the presence of the Cas9-D10A nicking nuclease, the two gRNAs induce single-stranded breaks on opposite strands of the DNA, creating a functional double-stranded break.
  • Pre-designed, unique CRISPR paired nickases are available for the coding regions of the human, mouse, and rat genomes (Paired Nickases Predesigns). Designs for any other regions or species are available as a custom request (see Custom CRISPR request form).
  • When ordering Sigma′s CRISPR paired nickases for a specific target, researchers will receive two separate ready-to-use, transfection-grade plasmids expressing gRNA from the human U6 promoter. The Cas9-D10A nicking nuclease must be purchased separately as either plasmid or mRNA.
Paired Cas9 Nickases

crispr-cas-3

Paired Cas9 Nickases - For optimal Cas9-D10A paired nickase functionality, guide RNAs should be designed in a 5’-to-5’ orientation with PAM spacing between 30 and 150 bp.


Purified RNA-only guide RNA

  • Ready-to-use gRNAs in a purified RNA format suitable for microinjection or cell culture.
  • Designed using the same stringent rules as our Sigma CRISPRs and CRISPR paired nickase plasmids.
  • The CRISPR RNA format is ideal for researchers creating transgenic animal models or for those concerned about genomic plasmid integration.
  • The RNA format avoids the need for a specific promoter, allowing for expression in most known cell types and organisms.
  • Wild type Cas9 (CAS9P) or Cas9-D10A nicking (CAS9D10AP) nuclease must be purchased separately as either plasmid or mRNA (CAS9MRNA, CAS9D10AMRNA).
  • Donor Designs available.


CRISPR Lentivirus

  • Sigma CRISPR Lentivirus for high efficiency knockout screens
  • The lenti format allows for efficient chromosomal integration of CRISPR components.
  • Transduce cells using Sigma’s unique single lentiviral CRISPR format, which features a Cas9 ORF flanked by puro and GFP elements, providing multiple options for monitoring stable cell populations.
  • Enables genome editing even in difficult to transfect cells.
  • Choose from our pre-designed CRISPR sites or submit custom targets
Sigma Lentiviral CRISPR

Sigma Lentiviral CRISPR: (A) Vector map of the all-in-one lenti CRISPR vector. (B) GFP signal post-transduction of HEK293 cells with particles made via pLV-U6g-EPCG. (C) HeLa cells were transduced with lentivirus (pLV-U6g-EPCG) followed by selection on puromycin and 6TG treatment to enrich for HPRT1 knockouts. Insertions and deletions were detected at the gRNA target site via mismatch assay (CEL-I). 


CRISPR positive and negative controls

CEL I image of CRISPR01 positive control (lane labeled EMX1s4+Cas9) and CRISPR02 positive control (lane labeled EMX1s4, EMX1as4+D10A)

CEL I image of CRISPR01 positive control (lane labeled EMX1s4+Cas9) and CRISPR02 positive control (lane labeled EMX1s4, EMX1as4+D10A)

CRISPR/Cas9 Vector

For additional information and to customize and purchase CRISPR click Order CRISPR below



Materials

     

References

Elitza Deltcheva, Krzysztof Chylinski, Cynthia M Sharma, Karine Gonzales, Yanjie Chao, Zaid A Pirzada, Maria R Eckert, Jörg Vogel, Emmanuelle Charpentier
Nature 2011-03-31
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the...Read More
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A Doudna, Emmanuelle Charpentier
Science 2012-08-17
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that ...Read More
Luciano A Marraffini, Erik J Sontheimer
Nature 2010-01-28
All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway. CRISPR loci are present in approximately 40% ...Read More
Le Cong, F Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D Hsu, Xuebing Wu, Wenyan Jiang, Luciano A Marraffini, Feng Zhang
Science 2013-02-15
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II...Read More
Prashant Mali, Luhan Yang, Kevin M Esvelt, John Aach, Marc Guell, James E DiCarlo, Julie E Norville, George M Church
Science 2013-02-15
Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) i...Read More
Haoyi Wang, Hui Yang, Chikdu S Shivalila, Meelad M Dawlaty, Albert W Cheng, Feng Zhang, Rudolf Jaenisch
Cell 2013-05-09
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demon...Read More