May 1, 2017 (Vol. 37, No. 9)
Generation of Microgram Quantities of sgRNA in Less Than One Hour
CRISPR is an acronym for Clustered Regularly Interspaced Palindromic Repeats, which are genomic loci found in many bacteria and archaea. The CRISPR/Cas9 pathway naturally allows for the elimination of genomic material from invading sources in bacteria, and has recently been adapted as a molecular biology tool to?edit genomes in a target-specific manner. Cas9 (CRISPR-associated protein 9) is a double- stranded DNA endonuclease that forms an active ribonucleoprotein (RNP) when complexed with guide RNAs (gRNAs) encoded at the CRISPR loci. gRNAs provide sequence-specificity to the RNP, directing the Cas9 nuclease to DNA targets resulting in double-strand breaks in the DNA.
In nature, S. pyogenes Cas9 is programmed with two separate RNAs, the CRISPR RNA (crRNA) and the transactivating crRNA (tracrRNA). The crRNA contains ~20 nucleotides of homology complementary to the strand of DNA opposite and upstream of the Protospacer Adjacent Motif (PAM) (NGG) sequence.
The tracrRNA contains partial complementary sequence to the crRNA, as well as the sequence and secondary structure that is recognized by Cas9. These sequences have been adapted for use in the lab by combining the tracrRNA and crRNA into one long single guide RNA (sgRNA)1 species capable of complexing with Cas9 to recognize the target DNA and induce double-strand DNA breaks (Figure 1).
Activation of the cellular double-strand?break machinery can lead to insertions and/or deletions (indels) through Non-Homologous End Joining (NHEJ), resulting in disruption of the gene at that specific locus. In the presence?of a homologous repair template, the homology-directed repair (HDR) pathway can be activated leading to the introduction of specific changes in the DNA at the targeted site.
This application note describes sgRNA synthesis using the new EnGen™ sgRNA Synthesis Kit, S. pyogenes, which simplifies the generation of custom sgRNA in an hour or less by combining template synthesis and transcription in a single-tube reaction.
Figure 1. An sgRNA is complexed with Cas9, S. pyogenes. Cleavage occurs three nucleotides upstream of the PAM sequence (red). sgRNAs are complementary to the strand opposite of the PAM.
Protocol
The EnGen sgRNA Synthesis Kit, S. pyogenes provides a simple and quick method for transcribing high yields of sgRNA in a single 30-minute reaction using the supplied reagents and target-specific DNA oligos designed by the user. This target-specific oligo contains a T7 promoter sequence, ~20 nucleotides of target-specific sequence, and a 14 nucleotide overlap region that anneals to a complementary region within the S. pyogenes Cas9-specific Scaffold Oligo (included in the EnGen 2X sgRNA Reaction Mix). The DNA polymerase within the Enzyme Mix extends both oligos from their 3′ ends creating a double-stranded DNA (dsDNA) template for transcription by T7 RNA Polymerase, also provided within the Enzyme Mix. Synthesis of the dsDNA template and transcription of RNA occur in a single reaction, resulting in the generation of a functional sgRNA.
Target sequences for modification are selected using online guide selection tools such as ChopChop or Desktop Genetics. Target-specific oligos to be ordered are designed using the EnGen sgRNA Template Oligo Designer within the NEBioCalculator® (NEBioCalculator.neb.com). Reactions are assembled at room temperature and incubated at 37°C for 30 minutes, followed by DNase I treatment. sgRNAs are purified using RNA spin columns, quantified by UV light absorbance and analyzed by gel electrophoresis. In vitro digestion of dsDNA templates in the presence of Cas9 is performed to demonstrate the functionality of sgRNAs synthesized using the EnGen sgRNA Synthesis Kit, S. pyogenes.
Results
Using the guidelines above, 72 target-specific oligos were designed and used to generate 72 different sgRNAs. Yields ranged from 4-45 μg, with most falling between 4-25 μg. sgRNA quality and length were evaluated by denaturing gel electrophoresis and SYBR Gold staining. The sgRNA control oligo synthesized with the EnGen sgRNA Synthesis Kit was incubated with Cas9, S. pyogenes to form an active RNP complex that cleaved a double-stranded DNA target in vitro.
Conclusion
The EnGen sgRNA Synthesis Kit, S. pyogenes can be used to generate microgram quantities?of functional custom sgRNAs in less than an hour. This method reduces protocol time with the single reaction-format and only requires the design of a single ~55 nucleotide ssDNA target-specific oligo for each sgRNA resulting, thus reducing the cost per reaction.
G. Brett Robb, Ph.D.([email protected]), is scientific director, RNA & Genome Editing at New England Biolabs.
Reference
1. Jinek, M. et al. (2012) Science 816-821. PubMed ID: 22745249
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