The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was discovered in bacteria, where it functions as an adaptive immune system against invading viral and plasmid DNA. In this system, short DNA sequences (spacers) from invading viruses are incorporated at CRISPR loci within the bacterial genome and serve as memory of previous infections. Reinfection triggers complementary mature CRISPR RNA (crRNA) to find a matching viral sequence. Together, the crRNA and transactivating crRNA (tracrRNA) guide CRISPR-associated (Cas) nuclease to cleave double-strand breaks in the corresponding foreign DNA sequences.1
The type II prokaryotic CRISPR “immune system” has been engineered to function as an RNA-guided genome-editing tool that is simple, easy, and quick to implement. Here, we describe two different recombinant Cas9 proteins: Cas9 (wild type SpCas9) and enhanced specificity Cas9 (eSpCas9), which has been shown to reduce off-target cleavage.2 These proteins can be combined with SygRNA® synthetic crRNAs and tracrRNAs to form ribonucleoprotein (RNP) complexes that target the specific genomic locus of interest (Figure 1).
Figure 1. Three Component CRISPR Cas9 System.The Cas9 ribonucleoprotein is made up of the Cas9 protein and a guide RNA, which can be divided into a tracrRNA and a crRNA. The crRNA is variable and complementary to the target of interest, while the tracrRNA sequence is static.
introduction of Cas9 RNP-sgRNA strengthens and expands the applications of CRISPR genome modification technology by eliminating the possibility of plasmid DNA integration into the host genome. This method also reduces risk for off-target effects due to the rapid degradation of the RNP after delivery; in many cases Cas9 RNP results in efficient genome modification with higher specificity when compared to cells transfected with Cas9 plasmid.1,3,4,5 This RNP technology has broad applications and has been shown to function in both mammalian and plant systems.6 Furthermore, Cas9 RNP delivery holds great promise for therapeutic applications including the recent successful generation of knock-in primary human T cells.7
One of the primary concerns with CRISPR is potential for off-target cleavage; eSpCas9 improves the specificity of the system by reducing these effects. The unwinding of target DNA by SpCas9 is driven by the sum of two forces: the positive charge of the chromosome-binding motif of the protein and the RNA:DNA interaction between the guide RNA and the target DNA. Therefore, weakening the binding efficiency of SpCas9 has the potential to increase the requirement for precise match between guide RNA and target DNA for unwinding of the target to occur.
To create eSpCas9, wild type SpCas9 was engineered to possess a relaxed binding efficiency, resulting in higher on-target fidelity without the loss of cleavage efficiency. To engineer this protein, alanine point mutations were made in the chromosome-binding motif of SpCas9.2 In testing eSpCas9 in combination with select gRNA (Figure 2), on-target cleavage efficiency was comparable to wild type SpCas9 with undetectable cleavage at select off-target sites.
Figure 2. eSpCas9 Reduces Off-Target Cleavage Compared with Cas9.In this experiment, K652 cells were nucleofected with SpCas9 or eSpCas9 and synthetic tracrRNA and EMX1-targeted crRNA. A CEL-1 assay showed equal cleavage efficiency between Cas9 and eSpCas9, while cleavage at a known off-target site7 was reduced when eSpCas9 was used compared to Cas9.
In general, the steps required for successful introduction of Cas9 RNP into cultured and primary cells are as follows:
This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
This lyophilized protein is shipped at ambient temperature. Once resuspended in the provided reconstitution solution, Cas9 and eSpCas9 are recommended to be stored at –20 °C.
Each kit contains the following components:
The following equipment and reagents are recommended but not provided in this kit:
CRISPR is amenable to a variety of transfection methods; therefore it is recommended to optimize methods to suit the cells of interest. We provide a variety of transfection reagents, cell culture media and plates, and custom DNA primers for detection of CRISPR-mediated genome editing. For your reference, we have suggested protocols below.
Approximately 18–24 hours before use, plate cells in complete growth medium. For most cell types, cultures should be 50–80% confluent at the time of transfection.
Microinjection protocols vary greatly depending on embryo type and researcher preferences. Microinjection of Cas9 RNPs has been demonstrated in the following organisms:
We present a new development in Cas9 technology for addressing the problem of CRISPR off-target effects. In addition, we provide detailed protocols for use of Cas9 RNP which has broad applications in both animal and plant models.
If cleavage is not observed, the following considerations may aid the researcher in troubleshooting:
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