Generating miR156 and miR157 loss-of-function mutations using the CRISPR-Cas9 genome editing

The use of T-DNA insertional mutations in our previous genetic analysis of miR156 and miR157 provided valuable information on how these miRNAs regulate vegetative phase change. However there are some caveats when working with T-DNA insertional

mutations. Firstly, T-DNA mutants don’t always work well with transgenic reporter lines as the transgenes are prone to silencing. When SPL-GUS reporters were crossed to

miR156a/c or miR157a/c mutants, we observed frequent occurrence of silencing. This might have been due to the increased SPL-GUS expression, or the common

components on the SPL-GUS construct and the T-DNA insertion causing RNAi. Using an rdr6 genetic background may help reduce the chance of silencing but will increase the difficulty of genetic analysis. The second complication brought by the T-DNA mutants is the fact that some T-DNA insertional mutations are from an ecotype other than Col. Even after several generations of introgression to Col, it is inevitable to have random chromosomal segments from another ecotype remaining in the line which could affect phase change phenotypes. Thirdly, the current T-DNA mutation library cannot guarantee a knock-out mutation in a given gene. In fact, when we were trying to identify miR156f

loss-of-function mutations, the T-DNA insertional mutation in miR156F resulted in increased transcript level of Pri-miR156F. Last but not least, T-DNA insertional mutations can cause chromosomal rearrangement such as translocations (Clark & Krysan, 2010), making certain lines harder to work with, and also increasing the difficulty establishing causal relationships between genes and phenotypes.

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In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic

Repeats)/Cas (CRISPR-associated) system brought significant advancement in targeted genome editing (Cong et al., 2013). The application of CRISPR-Cas9 system in

Arabidopsis for efficient genome editing has been successful (Yanfei Mao et al., 2013; Y. Mao et al., 2016). This system generates double-strand breaks (DSBs) at target sites mediated by the short guide RNA (sgRNA). DNA sequence modifications including insertion, deletion or point mutations are then achieved via the error-prone non- homologous end joining (NHEJ) pathway or sequence replacement through the error- free homologous recombination (HR) pathway. After stable mutations at the target loci are obtained, the CRISPR-Cas9 transgene can be removed by backcrossing.

CRISPR-Cas9 systems appear to be ideal for generating loss-of-function mutations in miR156 and miR157. There is a canonical PAM sequence (5’-NGG-3’) 4 nucleotides away from the 3’ of mature miR156 and miR157 in the Arabidopsis genome. When we design a guide RNA targeting this site, multiple miR156 or miR157 loci in the genome can be edited by this same CRISPR-Cas9 transgene. Since our goal is to find loss-of- function mutations, we can focus on screening deletion mutations resulting in the loss of mature miR156 or miR157 sequences in each locus. After a mutation in one of the miR156 or miR157 genes has been identified, we can cross it to WT to remove the CRISPR-Cas9 transgene, or harvest the seeds to allow mutations in other

miR156/miR157 loci to be induced. The ability to continuously induce loss-of-function mutations in different miR156/miR157 loci over generations is very helpful for generating double, triple or higher order mutants especially when some of the genes are linked.

With this rationale, we constructed CRISPR-Cas9 targeting miR156 or miR157 loci in

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transformants is achieved by seed fluorescence (Wu et al., 2015). Individual T2 plants were genotyped using primers flanking the miR156 mature sequences in different miR156 genomic loci. Seeds from mutant candidates were harvested individually and further screened/confirmed in the next generation. From the preliminary screening, we were able to identify mutations that resulted in the deletion of miR156 mature sequence in different miR156 loci, including a double mutant miR156e-1/miR156f-1. This is of particular interest since miR156E and miR156F are both located on chromosome 5. Figure 6.1 shows the nature of the miR156E and miR156F mutations. miR156e-1 is a mutation causing a 18-nucleotide deletion in miR156E genomic locus, which includes 15 nucleotides of the miR156 mature sequence and another 3 nucleotides right at the 3’ of it. miR156f-1 is a mutation causing a 79-nucleotide deletion in miR156F genomic locus, which includes the full miR156 mature sequence.

These preliminary results demonstrated that our CRISPR-Cas9 system is suitable for generating loss-of-function miR156/miR157 mutations in Col background. With additional work in future screening, we are likely to have a collection of high quality miR156/miR157 loss-of-function mutations.

104 Figure 6.1 CRISPR-Cas9 induced miR156 mutations.

Shown here are examples of CRISPR induced stable mutations in miR156E and miR156F. WT miR156E and miR156F sequences are aligned with homozygous mutations identified. Dashed lines represent deletions. Red colored nucleotides are mature miR156 sequences.