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CRISPR-Cpf1 expands genome editing to new target sites

CRISPR genome editing, facilitated by the CRISPR associated (Cas) enzyme, Cas9, is by far the most popular method for genome editing. It is relatively easy to purchase or generate the necessary CRISPR components to target most genes, and the technology can be implemented in cells ranging from bacterial cells to human cells. Cas9 is highly efficient at creating double-stranded breaks in DNA targets, especially when introduced into target cells bound to CRISPR RNAs, as a ribonucleoprotein (RNP) complex. However, Cas9 may not work for all genomic target sites. The main reason for this limitation is that Cas9 only recognizes cut sites that have a protospacer adjacent motif (PAM) of NGG. This PAM sequence must be present at the 3′ end of the desired target sequence for the RNP to bind. Thus, functional target sites may be limited or nonexistent in sequence regions or genomes with lower GC content.

Other Cas enzymes, that use different recognition sequences, have been found. Unfortunately, many of these are not suited for straightforward genome editing. Cpf1, however, has fairly good editing efficiency, and a PAM sequence of TTTV (where V is DNA base A, G, or C, but not T). IDT offers the Alt-R® CRISPR-Cpf1 System, which includes Cpf1 protein, and gRNA (Cpf1 does not require a separate tracrRNA). Watch the video recording from our webinar, Cpf1-based genome editing using ribonucleoprotein complexes, given by Dr Rolf Turk, to learn more about this alternative to Cas9 nuclease.

Also covered during the presentation is a brief comparison of the Alt-R CRISPR-Cpf1 System to our highly effective Alt-R CRISPR-Cas9 System.

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 recognizes an AT-rich PAM site, providing CRISPR target sites that are not available with the CRISPR-Cas9 System. In addition, Cpf1 nuclease 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 reagents

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

Alt-R® CRISPR-Cpf1 System

Alt-R® CRISPR-Cpf1 User Guide—ribonucleoprotein electroporation; Amaxa® Nucleofector® System—Get the protocol and understand the important considerations for delivery of ribonucleoprotein complexes to HEK-293 cells.

Alt-R® Genome Editing Detection Kit Protocol—Use this protocol with the Alt-R Genome Editing Detection Kit to determine on-target genome editing and estimate editing efficiency using T7 endonuclease I (T7EI).

Alt-R® CRISPR-Cas9 System

Getting started with Alt-R CRISPR-Cas9 genome editing
—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)—Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation.

CRISPR genome editing: 5 considerations for target site selection—Learn how your genome editing experiments can be improved with 5 quick tips for target selection and with reagents from the Alt-R CRISPR-Cas9 System

Author: Hans Packer, PhD, is a scientific writer at IDT.

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