Skeletal muscle spatial analyses have revealed unexpected regionalized gene expression patterns challenging the understanding of muscle as a homogeneous tissue. Here, we present a protocol for the spatial analysis of transcript and protein levels in murine skeletal muscle. We describe steps for tibialis anterior dissection, formaldehyde fixation, tissue chopper cutting, and hybridization chain reaction (HCR) detection and amplification. We then detail procedures for immunostaining, tissue clearing, and imaging. This protocol is easily adaptable to other tissues.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.