Using CRISPR knockin to express GFP fusion proteins in HEK-293 cells

purpose: knockin via CRISPR to introduce C-terminal GFP tag onto LAMP1 gene in the HEK-293 cell line

  • LAMP1 encodes LAMP1 lysosomal marker protein

CRISPR gene editing overview

  • found by Jennifer Doudna and Emmanuelle Charpentier

  • based on bacterial adaptive immune system in S. pyogenes

  • short DNA from invading bacteriophage incorporated into bacterial genome at CRISPR loci

    • allows bacteria to “remember”

    • upon reinfection Cas9 recognizes genome of bacteriophage

  • Cas9 is CRISPR associated nuclease 9

    • nuclease forms double stranded DNA breaks at precise points in the invading bacteriophage’s genome and destroys it

  • Cas9 nuclease is introduced along with a sgRNA

    • sgRNA base pairs with a 20 bp target sequence in the gene interest

    • once sgRNA is base paired, sgRNA will be recognized by Cas9

    • Cas9 generates DSB 3 base pairs upstream of PAM

  • after Cas9 cuts, DNA repair processes are responsible

    1. NHEJ - non homologous end joining pathway

      • leads to indels that disrupt target gene, a gene knockout

    2. HDR - homology-directed repair pathway

      • HDR requires donor repair template which allows insertion of DNA sequence, a gene knockin

MMEJ

  • MMEJ - microhomology-mediated end-joining to knockin GFP tag onto C-terminal end of LAMP1 gene

    • MMEJ shows some benefits in donor vector preparation

    • employs shorter donor homology arms