Home > CRO Services > AAV Vector Design > AAV-CRISPR Cloning

AAV-CRISPR Cloning

AAV-CRISPR Gene Editing

PackGene provides customized vector cloning services 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 SpCas9/SpCas9HF (A/B series), SaCas9/SaCas9HF (C/D series), AAV-CRISPR gene activation (E series), and NmCas9, AsCpf1, LbCpf1 (F/G/H series) respectively.

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, which is an endonuclease that cuts the double strain DNA. This speciality of recognition makes it feasiblt to find and bind to any desired sequence. Besides, researchers can introduce mutations where the cut area is repaired, or make precise nucleotide changes. 

Building suitable vectors are difficult or time-consuming when lacking of experience on vector operations, these difficulities can be solved through our professional team. We provides professional customized AAV-CRISPR vectors and Lentivirus-CRISPR vectors which are ready for experiments. 

Interested in our AAV services? Please contact us for a free consultation or  submit a request for a quotation.
SpCas9/SpCas9HF (A/B series)

SpCas9 is the first CRISPR 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, to activate gene transcription in the downstream position. The transcription level of activated genes can reach more than 1000 times.

Key properties

  • The use of an optimised sgRNA scaffold increases the cleavage efficiency of SpCas9HF by approximately 40%. SpCas9HF is a high fidelity version of SpCas9 that greatly reduces off-targeting and has no significant reduction in cleavage activity compared to wild type.
  • XA01 is an integrated vector with SpCas9 and sgRNA expression cassette in the same vector. The use of the H1 promoter (100 bp), which is smaller than the U6 promoter but not significantly less active, allows the sgRNA expression cassette to be placed in the same vector as SpCas9.
  • XA02 is a separate SpCas9 vector without a sgRNA expression cassette, which can be replaced with an EFS promoter of up to 410 bp as required.
  • XA03 to XA27 are U6-sgRNA expression cassettes with EFS or EF1 or CAG promoter-driven mCherry, Cre, mCherry-2A-Cre, RenillaLuciferase-2A-Cre, ZsGreen, EGFP, EGFP-KASH.
SaCas9/SaCas9HF (C/D series)

SaCas9 is a shorter version of Cas9 with similar activity to SpCas9, with a gene length of 3.2kb, as there is a more stringent PAM (NNGRRT), the frequency of PAM sequences in the gene, approximately once in every 32bp, and a target sequence length of 21nt-23nt, with a theoretically lower off-target rate than SpCas9.

Key properties

  • The use of an optimized sgRNA scaffold, significantly improves cleavage efficiency.
  • C1/D1 are integrated vectors for SaCas9HF with CMV promoter and sgRNA expression cassette.
  • C2/D2/C2XX/D2XX are separate SaCas9/SaCas9HF vectors without sgRNA expression cassette, which can be used with different vectors according to the experimental design.
AAV-CRISPR gene activation (E series)

Guided by a short RNA of 14-16 nt, wild-type SpCas9 binds to target sequence DNA but is unable to cleave and release it. At this point, the sgRNA backbone, which has been modified to form the MS2 RNA junction, can bind the MS2-P65-HSF1 transcriptional activation component, thereby activating gene transcription at downstream sites. The transcriptional level of the activated gene can reach more than 1000-fold.

NmCas9 is a shorter version of Cas9 with similar activity to SpCas9, with a gene length of 3.3 kb. Due to the recognition of a longer PAM (NNNNGATT), the frequency of PAM sequences in the gene, approximately once in every 128 bp, and a target sequence length of (21-23 nt), the theoretical off-target rate is much lower than for SpCas9, AsCpf1, LbCpf1 and LbCpf1. LbCpf1 possesses properties different from those of CRISPR-Cas9: (1) a shorter sgRNA backbone (2) recognition of the T-rich 5′ PAM (3) the target DNA is cut away from the PAM and produces a 5-base protruding sticky end.

NmCas9

The 3.3-kb-long NmCas9 is another alternative t version of Cas9 with similar activity to SpCas9. NmCas9 can be beneficial when the theorectical off- target rate is required to be much lower, due to the recognition of the longer PAM (NNNNGATT), the frequency of the PAM sequence appears around once every 128 bp, and the target sequence length is 21-23 nt.

  • Easy Access: Reliable database and library, stored with large variety of vectors, simply upload your sgRNA sequence and pick the vector from our library.
  • Time Saving: With our experts specilized in this area with more than 10 years’ experience, we are confident that  PackGene can carry out target design, map and vector production efficiently.
  • Expertise: To ensure the quality and maximize the use of our products,  PackGene recommends selecting 2-3 targets for non-validated gRNA.

Highlights
  • Optimized sgRNA scaffold increases 40% efficiency.
  • High-fidelity SpCas9HF can significantly reduce the 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 upon request.
  • XA03-XA27 belong to U6-sgRNA, involving mCherry, Cre, mCherry-2A-Cre, RenillaLuciferase-2A-Cre, ZsGreen, EGFP, EGFP-KASH, which are driven by EFS, EF1 or CAG promoter.
  • Vector library with all kinds of vectors applicable according to various demands without sgRNA expression.