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CRISPR genome editing

Use of the CRISPR (clustered regularly interspaced short palindromic repeats) and associated Cas9 enzyme for genome editing has been a major technological breakthrough, making genome modification in cells or organisms faster, more efficient, and more robust than previous genome editing methods. The Alt-R CRISPR-Cas9 System is an optimized genome editing solution that outperforms other CRISPR approaches for producing on-target, double-stranded DNA breaks.

We have also developed an alternative Alt-R CRISPR-Cas12a (Cpf1) System to open up CRISPR editing to additional areas in genomes.

The Alt-R CRISPR Systems were developed through comprehensive research on each component of the CRISPR-driven, double-stranded break generation critical for gene disruption and DNA insertion by homologous recombination.

Alt-R CRISPR-Cas9 System »

Efficient CRISPR reagents based on the commonly used Streptococcus pyogenes Cas9 system for lipofection or electroporation experiments. Protospacer adjacent motif (PAM) = NGG.

Alt-R CRISPR-Cas12a (Cpf1) System »

For additional target sites or for targeting AT-rich regions, use the Acidaminococcus sp. BV3LC CRISPR-Cas12a system in electroporation experiments. Protospacer adjacent motif (PAM) = TTTV. The new Alt-R Cas12a (Cpf1) Ultra also can recognize many TTTT PAM sites in addition to TTTV motifs, increasing target range for genome editing studies.

CRISPR nucleases »

Recombinant, high-purity Cas9 and Cas12a (Cpf1) endonucleases for genome editing experiments.

Genome editing detection »

T7 endonuclease I (T7EI) mismatch cleavage assay for detection of on-target editing, known off-target events, and estimation of genome editing efficiency in cultured cells.

Quick comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)

 Cas9 systemCas12a system
ApplicationsGeneral genome editing
  • For species with AT-rich genomes
  • For regions with limiting design space for use of the CRISPR-Cas9 system
Ribonucleoprotein components
  • gRNA options:
    1. crRNA and tracrRNA
    2. sgRNA
  • Cas9 endonuclease
  • crRNA
  • Cas12a endonuclease
Variants
  • Wild-type
  • HiFi
  • Nickases (H840A and D10A)
  • Wild-type
  • Ultra (Improved performance)
Cas9 crRNA:tracrRNA (option 1)

crRNA

  • Native: 42 nt
  • Alt-R: 35–36 nt (36 nt recommended)

tracrRNA

  • Native: 89 nt
  • Alt-R: 67 nt
Cas9 sgRNA (option 2)
  • Alt-R: 99–100 nt (100 nt recommended)
Cas12a crRNA
  • Native: 42–44 nt
  • Alt-R: 40–44 nt (41 nt recommended)
CRISPR enzyme
  • Class 2, Cas type II
  • M.W.*: 162,200 g/mol
  • Endonuclease domains: RuvC-like and HNH
  • Class 2, Cas type V
  • M.W.*: 156,400 g/mol
  • Endonuclease domain: RuvC-like only
DNA cleavage
  • Wild-type and HiFi: Blunt-ended cut 3 bases upstream of the protospacer sequence
  • D10A nickase with paired gRNAs: 5′ overhang
  • H840A nickase with paired gRNAs: 3′ overhang
  • PAM site often destroyed during genome editing
  • 5′ overhanging cut on the 5′ side of the protospacer sequence
  • PAM site may be preserved after genome editing
PAM sequenceNGG
  • TTTV for Cas12a V3
  • TTTN for Cas12a Ultra
Current recommendations for Alt-R RNP delivery
  • Lipid-mediated transfection
  • Electroporation (Alt-R enhancer recommended)
  • Microinjection
  • Electroporation (Alt-R enhancer recommended)
  • Microinjection

* Molecular weight of Alt-R nuclease
N = any base; V = A, C, or G

This comparison table is available for download (see page 2).

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