Analysis of endogenous protein localization, function, and dynamics is fundamental to the study
of all cells, including the diversity of cell types in the brain. However, current approaches are often low-throughput and resource-intensive. Here we describe a CRISPR/Cas9-based Homology independent Universal Genome Engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs AAV vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications, that can integrate into any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural circuit-specific protein modification, subcellular rerouting and sequestration, as well as truncation-based structure-function analysis. Thus, the “plug and play” nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

The complex skills underlying verbal and musical expression can be learned without external
punishment or reward, indicating their learning is internally guided. The neural mechanisms that
mediate internally guided learning are poorly understood, but a circuit comprising dopamine
releasing neurons in the midbrain ventral tegmental area (VTA) and their targets in the basal
ganglia (BG) are important to externally reinforced learning. Juvenile zebra finches copy a tutor
song in a process that is internally guided and, in adulthood, can learn to modify the fundamental
frequency (pitch) of a target syllable in response to external reinforcement with white noise. Here
we combined intersectional genetic ablation of VTA neurons, reversible blockade of dopamine receptors in the BG, and singing-triggered optogenetic stimulation of VTA terminals to establish
that a common VTA – BG circuit enables internally-guided song copying and externally reinforced syllable pitch learning.

Learning to vocalize depends on the ability to adaptively modify the temporal and spectral features of vocal elements. Neurons that convey motor-related signals to the auditory system are theorized
to facilitate vocal learning, but the identity and function of such neurons remain unknown. Here
we identify a previously unknown neuron type in the songbird brain that transmits vocal motor
signals to the auditory cortex. Genetically ablating these neurons in juveniles disrupted their
ability to imitate features of an adult tutor’s song. Ablating these neurons in adults had little effect
on previously learned songs, but interfered with their ability to adaptively modify the duration of
vocal elements and largely prevented the degradation of song’s temporal features normally caused by deafening. These findings identify a motor to auditory circuit essential to vocal imitation and to the adaptive modification of vocal timing.