AAV-CIRSPR Cloning

AAV-CRISPR Gene Editing

PackGene provides customized vector cloning for CRISPR gene editing. The knockout and repair of AAV-CRISPR vectors are divided into 4 categories for in vivo experiments on genetically edited animals, which are respectively SpCas9/SpCas9HF (A/B series), SaCas9/ SaCas9HF (C/D series), AAV-CRISPR gene activation (E series), and NmCas9, AsCpf1, LbCpf1 (F/G/H series).
CRISPR is a state-of-the-art tool for gene editing. Because of its powerful performance and relatively simple operation, researchers are carrying out increasing number of CRISPR experiment
CRISPR is consists of two parts, gRNA and Cas9. gRNA can recognize a particular sequence of DNA and recruit the Cas9. Cas9 is an endonuclease that cuts the double strain DNA. Through specially design gRNA, Cas9 can find and bind to any desired sequence. CRISPR plays multiple roles in gene editing. Researchers can introduce mutations where the cut area is repaired, or make precise nucleotide changes.
Building suitable vectors would be difficult or time-consuming for certain laboratories. PackGene provide professional customized AAV-CRISPR vectors and Lentivirus-CRISPR vectors which are ready for experiments.

Interested in our AAV services?   Contact us for a free consultation or Request a quote.

SpCas9

SpCas9 is the first CIRSPR nuclease to be identified and the PAM sequence is NGG. The PAM sequence is around 4.2kb in length and it appears about once every 8bp. PackGene applies an EFS (EF1a short version) promoter less than 300 bp or a miniCMV promoter (180 bp) to drive SpCas9, so that the total sequence length would not exceed the AAV length limit of 5Kb. Under the guidance of 14-16 nt short RNA, wild-type SpCas9 can bind to the target sequence DNA while it cannot be cleaved and released. Meanwhile, the sgRNA backbone with a modified MS2 RNA linker can bind to the MS2-P65-HSF1 transcriptional activation component, so as to activate gene transcription in the downstream position. The transcription level of activated genes can reach more than 1000 times.

SaCas9

3.2kb in length, SaCas9 is a short version of Cas9 with similar activity to SpCas9. Because of the stricter PAM(NNGRRT), PAM sequence appears around once in every 32bp. As the length of target sequence is 21nt-23nt and less frequently appear in the genome, the off-target rate is lower than SpCas9 theoretically. Moreover, the smaller size of SaCas9 could better fit itself into the AAV vectors.

NmCas9

The 3.3-kb-long NmCas9 is a short version of Cas9 with similar activity compared with SpCas9. Due to the recognition of the longer PAM (NNNNGATT), the frequency of PAM sequence appears around once every 128 bp, and the target sequence length is 21-23 nt, the theoretical off-target rate is much lower than SpCas9.

PackGene Superiority

  • Easy Access: simply upload your sgRNA sequence and pick the vector from our library.
  • Time Saving: PackGene carries out target design, map and vector production efficiently.
  • Professional: PackGene recommends selecting 2-3 targets for non-validated gRNA

Highlights

  • Optimized sgRNA scaffold increases 40% efficiency
  • High-fidelity SpCas9HF can significantly reduce off-target effect and maintain cleavage activity
  • sgRNA and SpCas9 can be fit in the same vector with the application of a smaller H1 promoter.
  • EFS promoter can be replaced with one below 410bp as needed
  • XA03-XA27 belong to U6-sgRNA, involving mCherry、CremCherry-2A-CreRenillaLuciferase-2A-CreZsGreenEGFPEGFP-KASH, which are driven by EFS, EF1 or CAG promoter.
  • Different vectors can be used according to various demands without sgRNA expression
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