Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Abstract

The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.

Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction

“…However, recent developments in CRISPR genome editing have created new opportunities for generating cell culture models without first generating mutant mice. Several studies including ours demonstrated that clock genes can be knocked out efficiently in culture using CRISPR ( 55 – 59 ). One key limitation in cell models today is the lack of precise endogenous phase and period reporters working like clock hands other than the mPer2-Luc reporter in MEFs.…”

Quantitative comparison of CRISPR-Cas9-mediated mutation efficiency between mice and MEFs using digital PCR assays

“…Before we initiated the project in mouse models, feasibility of the project was tested in a clock cell model U2OS which has been proven to have a functional clock and to be amenable to CRISPR genome editing 17, 47, 52 . When the motifs harboring W448 in PER1 and W419 in PER2 were targeted by CRISPR, diverse indel mutations were generated including in-frame mutations leading to deletion of several AAs (Fig S2).…”

“…Recent developments in genome editing have created new opportunities for generating cell culture models without first generating mutant mice. Several studies including ours demonstrated that clock genes can be knocked out efficiently in culture using CRISPR 17,[44][45][46][47] . To our knowledge, however, there are no HDRmediated mutations made in clock genes in MEFs by CRISPR.…”

Endogenous circadian reporters reveal critical consequences of diverse novel mutations in clock genes

“…However, recent developments in CRISPR genome editing have created new opportunities for generating cell culture models without rst generating mutant mice. Several studies including ours demonstrated that clock genes can be knocked out e ciently in culture using CRISPR [55][56][57][58][59] . One key limitation in cell models today is the lack of precise endogenous phase and period reporters working like clock hands other than the mPer2-Luc reporter in MEFs.…”