Genome Editing
Support and Educational Content

No Cas9 PAM NGG sequence in your target region for genome editing?

Occasionally, researchers performing genome editing are confronted with a narrow target region that lacks the recommended Cas9 NGG protospacer adjacent motif (PAM). S. pyogenes Cas9 endonuclease, the enzyme most commonly used for these experiments, does not effectively recognize target sequences lacking an NGG [1]. While Jiang et al. have shown that S. pyogenes Cas9 can also recognize NAG, it does so less efficiently [2].

The Acidaminococcus sp. BV3LC Cpf1 enzyme uses a PAM site of TTTV (i.e., TTTA, TTTC, TTTG). It is therefore, especially useful for targeting AT-rich regions. Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cpf1 nuclease (Figure 1). We, therefore, strongly recommend using positive control crRNAs to establish that your cells can be edited by Cpf1. In addition, we recommend testing 3 or more crRNAs per target gene.

Cpf1 PAM site is TTTV (i.e., TTTA, TTTC, TTTG)

Figure 1. Maximize successful CRISPR-Cpf1 genome editing by using TTTA, TTTC, or TTTG as the PAM site. HEK-293 cells were transfected with ribonucleoprotein (RNP: Alt-R® A.s. Cpf1 Nuclease 2 NLS complexed with Alt-R CRISPR-Cpf1 crRNA; IDT) as instructed in the Alt-R CRISPR-Cpf1 User Guide—RNP electroporation, Amaxa® Nucleofector® system (available at www.idtdna.com/CRISPR-Cpf1). TTTN sites from 6 genes were used to design 232 Alt-R CRISPR-Cpf1 crRNAs. Editing efficiency was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required for T7EI endonuclease assays. PAM = protospacer adjacent motif (Cpf1 PAM sequence is TTTV, where V = A, C, or G); N = any base


The A.s. Cpf1 nuclease is provided by IDT as part of the Alt-R® CRISPR-Cpf1 System, for use in electroporation experiments. We recommend using the Alt-R A.s. Cpf1 Nuclease 2NLS combined with the Alt-R CRISPR-Cpf1 crRNA to generate a ribonucleoprotein (RNP) editing complex. The Alt-R Cpf1 Electroporation Enhancer is critical for optimal delivery by electroporation and is recommended for all experiments using this transfection method. View the Alt-R® CRISPR-Cpf1 System User Guides for guidance on electroporation and delivery of the RNP into your cell lines. 

 

In addition, Cas9 from S. thermophiles recognizes the PAM sequences NNAGAA and NGGNG [3, 4], while Cas9 from N. meningitidis recognizes NNNNGATT and NNNNGCTT, albeit the latter enzyme requires a 24 nt protospacer [5].

Keep these options in mind when selecting crRNA protospacer sequences.

Learn more about the Alt-R CRISPR Systems at www.idtdna.com/CRISPR

References

  1. Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827–832.
  2. Jiang et al. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 31:233–239.
  3. Esvelt KM, Mali P, et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 10(11):1116–1121.
  4. Ran FA, Hsu PD, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11):2281–2308.
  5. Hou Z, et al. (2013) Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA 110:15644–15649.

Product focus—genome editing with Alt-R® CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Additional reading

Designing sgRNAs for CRISPR-Cas9 experiments—This general background article describes features of CRISPR crRNA protospacer and trRNA sequences used in genome editing experiments.

5 pieces of data that will change how you set up your CRISPR-Cas9 experiments—To improve the efficiency of CRISPR-Cas9 genome editing, IDT scientists evaluated several factors that influence how we design and perform genome editing experiments. Review the data and results for 5 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

Consistent, high performance genome editing—Product spotlight: Looking to improve the performance of your CRISPR-Cas9 genome editing application? The Alt-R® CRISPR-Cas9 System offers potent on-target editing, easy implementation, and reduced cellular toxicity.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


CRISPR-Cas9 Genome Editing

With Alt-R CRISPR-Cas9 System get the core components for improved on-target editing performance, while experiencing lower toxicity, and no innate cellular response.

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Related Content

Product page:

Alt-R CRISPR-Cas9 System

Webinar:

New RNA tools for optimized CRISPR/Cas9 genome editing

Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Poster:

Quantitative Measurement of CRISPR/Cas9 Gene Editing at the Level of Genomic DNA for sgRNA Site Selection Algorithm Development

User guide:

Alt-R CRISPR-Cas9 System User Guide