Gene Editing in Chlamydomonas Using the SCREAM Technique

In the model alga Chlamydomonas reinhardtii, CRISPR (clustered regularly interspaced short palindromic repeat)-based gene editing using Cas (CRISPR-associated) enzymes enables both (a) insertion of large gene cassettes and (b) the creation of knockouts based on the introduction of indels, and specific mutations via mutation-directing oligonucleotides. Owing to the relatively low efficiency of this process, selection markers are frequently used to enrich the candidate pool prior to screening, which typically employs PCR. Unfortunately, few selection markers are available for Chlamydomonas. Furthermore, each marker requires different selection media, and deletion of the selectable marker can be difficult. When multiple successive gene editing steps are required, the use of these markers becomes onerous. The SCREAM (sequential CRISPR via recycling endogenous auxotrophic markers) technique employs an endogenous gene as a marker, the mutation of which can be selected both in the forward (loss of function) and reverse (gain of function) directions. During the first gene editing step, crRNA and mutation-directing oligonucleotides are provided for both the marker and the first target gene (Target 1). Candidates with edited marker genes are selected by loss of marker function, prior to screening for the desired modification of the first target gene. Using a successful candidate, a subsequent gene editing step directs reversion of the mutant marker gene to wild-type status, with candidates being selected on auxotrophic media to detect the regain of function of the auxotrophic marker to wild type (i.e., reversion). Simultaneously, a second target gene modification is produced using Target 2-specific crRNA and oligonucleotides. Revertants, now with a wild-type auxotrophic marker, are then screened for the specific mutation of Target 2. This reversion strategy enables a single selectable marker to be reused indefinitely, facilitating the creation of many successive mutations in a single cell line. As the marker can be completely reconstituted, strains can be created in which only the target gene is altered. Employment of homology-directed repair, using single-stranded oligonucleotides for mutation creation, enables the creation of site-directed mutants, tag insertion, and gene knockouts or reversion, rather than the insertion of large gene cassettes. In this implementation, nitrate reductase is used as the endogenous auxotrophic marker, and the adenine phosphoribosyltransferase gene is used as an example of a target gene. Key features • A single endogenous selectable marker gene is used for all successive mutational steps, employing only two standardized selection media. • An indefinite series of gene edits created in a single cell line enables functional analysis of redundant gene families. • Suitable applications include gene tagging, gene knockout, restoration of mutated genes to wild type, and site-directed mutagenesis to study gene function. • Widely applicable to existing cell lines.