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Novel CRISPR-Cas12a Orthologs Expand Gene Editing Toolkit for Plants


NEW YORK – Researchers in the US and China have compared the genome editing efficiency of several orthologs of the CRISPR nuclease Cas12a, establishing an expanded toolbox for efficient multiplexed genome engineering with improved target accessibility and scalability in plants.

In a paper published on Monday in Nature Communications, the researchers said that even though Cas12a has been widely used and engineered for genetic manipulation in mammalian cells, only three Cas12a orthologs — LbCas12a, AsCas12a, and FnCas12a — have been used in plants to date. LbCas12a is most popular due to its high editing activity in rice, maize, Arabidopsis, tomato, Nicotiana benthamiana (a tobacco relative), lettuce, cotton, and citrus. FnCas12a is also an efficient editor in plants.

Plant genome editing can be used to accelerate crop breeding, but Cas12a specifically can allow for simultaneous modification of many genes in elite cultivars, which may be difficult or impractical to achieve with conventional breeding or genetic engineering methods. However, the researchers said, the TTTV protospacer adjacent motif (PAM) requirement of Cas12a limits its targeting scope. And although many multiplexing strategies have been developed in plants using Cas9 and Cas12a, a system that can achieve highly efficient biallelic editing at multiple targets has not yet been demonstrated. Without high biallelic editing efficiency, it's impractical to obtain multigene knockouts in crops through subsequent breeding, they added.

To address these problems, the researchers screened nine Cas12a orthologs that hadn't been used in plants before and identified six — ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a, and MbCas12a — that possessed high editing activity in rice.

Specifically, Mb2Cas12a stood out as having high editing efficiency and tolerance to low temperature. Further, the researchers said, an engineered Mb2Cas12a-RVRR variant enabled editing with more relaxed PAM requirements in rice, yielding genome coverage that was two times higher than with the wild-type Streptococcus pyogenes Cas9 (SpCas9).

To enable large-scale genome engineering, the researchers then compared 12 multiplexed Cas12a systems and identified a system with nearly 100 percent biallelic editing efficiency that was able to target as many as 16 sites in rice — the highest level of multiplex editing in plants to date using Cas12a. They then developed two compact single-transcript Cas12a interference systems for multi-gene repression in rice and Arabidopsis.

To showcase the Mb2Cas12a variants and the highly multiplexed Cas12a system, the researchers performed multiplexed editing of six sites on five genes to engineer disease resistance and yield certain traits simultaneously in rice, noted corresponding author Yiping Qi, an associate professor of plant science and landscape architecture at the University of Maryland.

He said his team used Mb2Cas12a because it was compatible with a relaxed PAM site in their target gene. "We could not use the widely used LbCas12a or AsCas12a for this because they would not recognize that PAM site," Qi added.

The researchers began by evaluating the nine Cas12a orthologs in rice protoplasts and found that  eight showed a preference for TTV PAMs in vitro. In addition, they tested ErCas12a44, which was developed by genome engineering firm Inscripta, which named it MAD7.

Only Mb2Cas12a had an editing efficiency of about 10 percent at two relaxed VTTV PAM sites. When they tested it at 18 target sites, they found that the nuclease could efficiently edit 13 with mutation frequencies of approximately 15 percent or greater, and could reliably generate stable knockouts of two target genes at VTTV PAM sites.

The researchers then engineered variants of four orthologs, including Mb2Cas12a, to test their editing efficiencies at several other PAM sites. All four variants could edit the TATA PAM sites with higher average editing efficiencies than the control variant, but only the Mb2Cas12a variant could edit the two TATC PAM sites. It also had high editing frequencies at all TATV PAM sites, outperforming all other variants.

To further broaden the target range of Mb2Cas12a, the researchers then engineered a second variant and tested its editing activity at 51 targeting sites with canonical and altered PAMs in rice protoplasts. This variant showed editing activities at six PAM sites, significantly broadening Cas12a's target range.

The researchers then moved on to compare 12 multiplexed Cas12a systems for high-efficiency large-scale biallelic genome editing and found system B to be the best. They then used this system to simultaneously edit five trait genes to enhance yield and blight disease resistance in rice.

The study, which provides a new "Cas12a toolbox" for plant genome engineering, is likely to be greeted with satisfaction by genome editing researchers. Kan Wang, a scientist at the Crop Bioengineering Center at Iowa State University, noted that she'd heard of the Qi's work but hadn't yet read the paper. "I know [this] will be very useful because they are expanding the scope for editing, hence give us much more possibilities and flexibility in changing a gene," she said.

Qi also noted that the multiplexed Cas12a expression system should have implications for mammalian biology, and that similar strategies "would likely enhance multiplexed editing in non-plant systems, as well."

Broad Institute researcher John Doench, who studies CRISPR for human biology and participated in a study last July to optimize Cas12a for pooled genetic screening, added that comparisons like the one undertaken by Qi and his team are also valuable for the CRISPR research community as a whole.

"Rigorous and comprehensive comparisons are enormously helpful for enabling researchers who might be otherwise paralyzed by an abundance of options," he said.