A quick-to-implement and optimized CRISPR-Cas9 protocol to obtain insertional and small indel mutants in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii is a leading model organism in algal research, widely used to study photosynthesis, chloroplast and cilia biology, and more recently, metabolism, light signaling, the cell cycle, and algal biotechnology. Its sequenced genome has significantly accelerated research in the field, while improved genome-editing tools are key to advancing reverse genetics and genetic engineering. Building on previous advances, we present a streamlined and efficient CRISPR-Cas9 protocol for generating knockout mutants in Chlamydomonas via non-homologous end joining (NHEJ), using only commercially available reagents. Additionally, we introduce a cost-effective, PCR-based screening method capable of detecting mutants with large insertions as well as short indels -as small as one base pair- thereby enhancing overall CRISPR efficiency.•This protocol is easy to setup and can be fully executed using commercially available reagents.•This protocol allows for quick implementation and generation of mutants: 5 weeks from design to sequencing of candidate mutants.•This protocol describes a novel PCR-based strategy to identify mutants containing short indels. Screening is designed to identify large insertion mutants and the often overlooked small indel mutants.

Keywords: CRISPR gene editing and small indel detection in Chlamydomonas; CRISPR-Cas9 protocol; Chlamydomonas; Indel detection, knockout mutants; Microalgae; Simple and quick.

Graphical abstract

Fig. 1

CRISPR-Cas9 design to target PSY1 gene. Scaled representation of the PSY1 gene is shown with its five exons (black boxes, E1–5). A gRNA was designed in the beginning of the first exon of this gene: the sequence is shown below (light blue), with the predicted cut site by the Cas9 (triangle) and the PAM region (grey). In this example, 4 screening primers were designed, two for the screening of large insertions (SLfw and SLrv, red) and two for the screening of short indels (SID). SID primers are both reverse primers which are paired with SLfw to detect short indels by PCR: SIDrv (light blue) features the same sequence as the gRNA, while SIDrv* (black) features the 3 bases at 3′ changed (light red), and serves as a negative control, to check that mismatches caused by the introduction of short indels at this locus do not result in PCR amplification. For the in vitro test a single forward primer was designed (I.V.fw, blue), and SLrv was employed as reverse primer for this test.

Fig. 2

PCR test of all the designed primers for PSY1. All PCR reactions were performed at 64 °C and yielded bands with the length indicated on top or no band (NA), as expected. A) PCR for the screening of large insertions (SLfw+SLrv), B) PCR for the screening of short indels (SLfw+SIDrv), C) negative control of short indels screening (SLfw+SIDrv*), D) in vitro test PCR (I.V.fw+SLrv). Two technical replicates are shown for each amplicon. Uncropped electrophoresis gel images including the DNA ladder are provided in Supplemental Fig. 1.

Fig. 3

In vitro test of CRISPR machinery designed to target PSY1. PCR was performed as indicated in Fig. 1 (I.V.fw+SLrv) and yielded amplicons of 769 bp; these were afterwards purified (left electrophoresis gel). In vitro digestion gave the two expected fragments of 473 and 296 bp (cleaved, right electrophoresis gel). The additional largest band corresponds to rests of the original undigested (uncleaved) amplicons. Uncropped electrophoresis gel images including the DNA ladder are provided in Supplemental Fig. 2.

Fig. 4

General overview of the CRISPR design (Step 1–3). Step 1.1. Design gRNA(s) for your gene of interest (GOI), but do not purchase gRNA(s) before successfully testing all primers. Step 1.2 Design all the primers required for this protocol, which will depend on the target site of the chosen gRNA(s): screening primers (SLfw, SLrv), indel primers (SIDrv, SIDrv*) and in vitro primers (I.V.fw, I.V.rv). Step 2. Test the designed primers (Step 2), by performing 4 different PCRs with gDNA from the strain to be used for CRISPR (follow indications in Table 1). If PCRs give the expected bands then purchase the designed gRNA(s); if not, go back to Step 1.1 and design new gRNA(s). Step 3. Test your RNPs (in vitro test): for this purpose, perform a PCR using I.V. primers and purify the resulting amplicon; also, assemble RNPs as indicated in “Assembly of the CRISPR-Cas9 machinery” following iv (in vitro test) reagent proportions; finally, in vitro digest the purified amplicon with the assembled RNPs, purify the reaction and perform an electrophoresis run. PSY1, as described here, can be used as positive control in Step 3.

Fig. 5

A. Screening of psy1 mutants by phenotype: psy1 mutants exhibit pale green coloration. B. Screening of psy1 mutants by genotype was carried out using primers to detect large insertions (SLfw+SLrv, giving L band) and short indels (SLfw+SIDrv, giving ID band). Screening by genotype resulted in three possible scenarios: a) wt colonies displayed both L and ID bands with the length expected in the absence of mutations at the cut site; b) large insertion mutants displayed L bands with much higher length; c) short indel mutants displayed the same L band with the same size as the wt, but didn’t display the ID band. C. Screening and sequencing of psy1 mutants from the latter scenario, one with a short insertion (in) and one with a short deletion (del). Uncropped electrophoresis gel images including the DNA ladder are provided in Supplemental Fig. 3.