Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

Automated DNA sequencing generates large volumes of genomic sequence data relatively rapidly. Many genetic sequences are discovered in advance of other, less easily obtained, biological information. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism. Reverse genetics systems can also allow the recovery and generation of infectious or defective viruses with desired mutations. This allows the ability to study the virus in vitro and in vivo.

In order to learn the influence a sequence has on phenotype, or to discover its biological function, researchers can engineer a change or disrupt the DNA. After this change has been made a researcher can look for the effect of such alterations in the whole organism. There are several different methods of reverse genetics:

Site-directed mutagenesis is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle codon changes in the open reading frame to identify important amino residues for protein function.^{[citation needed]}

Alternatively, the technique can be used to create null alleles so that the gene is not functional. For example, deletion of a gene by gene targeting (gene knockout) can be done in some organisms, such as yeast, mice and moss. Unique among plants, in Physcomitrella patens, gene knockout via homologous recombination to create knockout moss (see figure) is nearly as efficient as in yeast. In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome. In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs. In gene knock-in, the endogenous exon is replaced by an altered sequence of interest.

In some cases conditional alleles can be used so that the gene has normal function until the conditional allele is activated. This might entail 'knocking in' recombinase sites (such as lox or frt sites) that will cause a deletion at the gene of interest when a specific recombinase (such as CRE, FLP) is induced. Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted to a specific subset of tissues.^{[citation needed]}

Another technique that can be used is TILLING. This is a method that combines a standard and efficient technique of mutagenesis with a chemical mutagen such as ethyl methanesulfonate (EMS) with a sensitive DNA-screening technique that identifies point mutations in a target gene.^{[citation needed]}

In the field of virology, reverse-genetics techniques can be used to recover full-length infectious viruses with desired mutations or insertions in the viral genomes or in specific virus genes. Technologies that allow these manipulations include circular polymerase extension reaction (CPER) which was first used to generate infectious cDNA for Kunjin virus a close relative of West Nile virus. CPER has also been successfully utilised to generate a range of positive-sense RNA viruses such as SARS-CoV-2, the causative agent of COVID-19.