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Fragment Deleted Stable Cell Line Generation


CRISPR/Cas9 has been widely utilized to facilitate functional knockout of protein coding genes...

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Fragment Deleted Stable Cell Line Generation

CRISPR/Cas9 has been widely utilized to facilitate functional knockout of protein coding genes. However, a single site of gene editing may not be sufficient to disrupt protein’s structure (for example, miRNA stem-loop) and function. To overcome this potential limitation, paired guide RNAs targeting both 5’ and 3’ of the target region have been shown to result in larger deletions, thus resulting in loss-of-function completely.

Dual sgRNA-directed large gene deletion mediated by CRISPR/Cas9 system is a robust tool for generating deletions of long non-coding RNAs (lncRNAs), MicroRNAs (miRNAs) and regulatory sequences [1,2]. Several recent studies have reported the CRISPR/ Cas9 system-induced large genomic deletion, ranged from 23kb to 1Mb, in mouse [3], C. elegans [4], rabbit [5] and human cell lines [6].

Creative Biogene is experienced in producing a fragment deleted cell line using any mammalian cell line and targeting sequences of your interest. Utilization of two sgRNAs targeting the immediate sequence upstream and downstream is a viable method for functional knockout of a target protein completely.

Fragment Deleted Stable Cell Line Generation

Our services include:

  1. Pre-determined easy to transfect cell lines
  2. Design and construct sgRNA vectors targeting both 5' and 3' of the target region
  3. Co-transfected into cells
  4. Clonal screening and selection
  5. Validation of knockout by Sanger sequencing
  6. Microbial/sterility testing

Applications:

  1. Functional miRNA knockout
  2. Completely lncRNA knockout
  3. Long-term studies of regulatory sequences

References:

  1. Han J, Zhang J, Chen L, et al. Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA biology, 2014, 11(7): 829-835.
  2. Chen X, Xu F, Zhu C, et al. Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans. Scientific reports, 2014, 4: 7581.
  3. Zhou J, Wang J, Shen B, et al. Dual sgRNAs facilitate CRISPR/Cas9‐mediated mouse genome targeting. The FEBS journal, 2014, 281(7): 1717-1725.
  4. Song Y, Yuan L, Wang Y, et al. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system. Cellular and molecular life sciences, 2016, 73(15): 2959-2968.
  5. Shah R R, Cholewa-Waclaw J, Davies F C J, et al. Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders. Wellcome open research, 2016, 1.

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