CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci

Abstract

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in

“…Recently, CRISPR/Cas9 technology has been developed to edit and label genomic loci in the living cell due to its accuracy and efficiency on the recognition of DNA and RNA (Nelles et al, 2016 ; Shao et al, 2016 ; Qin et al, 2017 ; Tasan et al, 2018 ). CRISPR/Cas9-mediated gene KI usually employs donor templates with long homologous arms, ranging from dozens of nucleotides for single-stranded oligodeoxynucleotides (ssODNs) to several hundred base pairs for larger fragments.…”

“…Primers (IGE Biotechnology) contain 20 nt micro-homology arms (MHAs) around the cleave site (3 nt upstream of PAM) and 25 to 30 nt overlapping region of the universal template plasmid. In addition, all primers were 5′ thiophosphorylated to enhance the half-life of linear donor fragments as previously reported (Orlando et al, 2010 ; Tasan et al, 2018 ) and further lengthen the phosphorothioate bonds to their last 5 nt of 5′ end. PCR was performed using KOD FX (TOYOBO) with a step-down cycle condition following manufacturer’s instructions.…”

“…Once the clones grown in 12-well plates reached ~80% confluency, half of the cells were collected for genomic DNA (gDNA) extraction (Qiagen) and the other cells were preserved in liquid nitrogen until further use. Genotyping and Sanger DNA sequencing were carried out as described previously (Tasan et al, 2018 ). Briefly, all the genotyping PCRs were performed using KOD FX polymerase (TOYOBO) according to the manufacturer’s protocol.…”

Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag

“…Our CRISPR-Tags are consisted of no more than six repeats (24 CRISPR target sites). For comparison, a recent paper demonstrated an optimized TetO repeat for labeling endogenous loci while emphasizing that 96 repeats of TetO was required for long-term imaging 25 . The small size and low-repetitiveness of our CRISPR Tags are advantageous because they are less likely to perturb the chromatin structure or affect transcription and replication.…”

“…One limit of CRISPR-Tag strategy is its use for tagging regulatory elements, such as enhancers, which is a general problem for all DNA tags. TetO repeats were integrated 16 kb far away from target genes 25 . ANCH tag was inserted adjacent to the promoter of the transgene 24 .…”