Patent Application
20230183783
A variety of clustered regularly interspaced short palindromic repeats—CRISPR-associated (“Cas”) proteins have been discovered to have a collateral cleavage activity useful in detection (e.g., diagnostic) systems to detect particular nucleic acids of interest. See, for example, review by Sashital Genome Med 2018:10, 32.
The present disclosure provides improved detection (e.g., diagnostic) technologies that utilize Cas-protein collateral activity.
Among other things, the present disclosure identifies the source of a problem with use of certain Cas enzymes in certain collateral activity assays. For example, the present disclosure documents that certain such assays include a step that involves incubation at an elevated temperature for a period of time, and various Cas enzymes may be insufficiently stable to maintain a sufficient level of activity (e.g., collateral activity) under such conditions. In many embodiments, such a step may be or comprise a nucleic acid extension and/or amplification step.
Alternatively or additionally, the present disclosure provides the insight that particularly desirable embodiments of various collateral activity assays are those that can be performed in a single reaction vessel (i.e., so-called “one pot”) assays. The present disclosure appreciates that Cas enzymes whose activity (e.g., collateral cleavage activity) is insufficiently stable to maintain sufficient activity through any and all elevated-temperature step(s) (which may be or include, for example, one or more nucleic acid extension and/or amplification step(s)) may not be useful in such one-pot assays. The present disclosure furthermore documents that certain Cas protein(s) (e.g., Cas13 and Cas12) are insufficiently stable at relevant temperature(s), e.g., at temperatures at which nucleic acid extension and/or amplification reactions are typically performed (e.g., above about 60-65° C.).
The present disclosure encompasses the recognition that thermostable variants of various Cas proteins (e.g., Cas9) have already been described and/or otherwise made publicly available (see, for example, Mougiakos et al. Nat Commun. 8:1647, 2017). Those skilled in the art are able to compare such thermostable variants with related non-thermostable homologs (e.g., orthologs), in order to assess sequence changes and/or elements that may be necessary and/or sufficient to achieve thermostability, and furthermore can identify such sequence changes and/or elements in other homologs (e.g., orthologs) and/or can introduce them thereinto. Still further, those skilled in the art are well aware of potential sources of naturally-occurring thermostable Cas proteins (e.g., in microbes that survive in elevated temperature conditions, such as in sea vents, or are otherwise thermophilic). Thus, those skilled in the art, reading the present disclosure, could readily identify and/or develop appropriate thermostable Cas proteins for use as described herein.
In some embodiments, a useful thermostable Cas protein is a Cas12 or Cas13 homolog (e.g., ortholog). In some embodiments, a useful thermostable Cas protein is a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99% or 100% sequence identity to any one of SEQ ID Nos. 1-283.
Alternatively or additionally, in some embodiments, a useful thermostable Cas protein performs (e.g., its collateral cleavage activity functions sufficiently) at temperatures above about 50° C.; in some embodiments, above a temperature selected from the group consisting of about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., about 80° C., about 81° C., about 82° C., about 83° C., about 84° C., about 85° C., about 86° C., about 87° C., about 88° C., about 89° C., about 90° C., about 91° C., about 92° C., about 93° C., about 94° C., about 95° C., about 96° C., about 97° C., about 98° C., about 99° C., about 100° C., or combinations thereof. In many embodiments, useful thermostable Cas protein performs (e.g., its collateral cleavage activity functions sufficiently) at temperatures above about 60° C.
In some embodiments, a useful thermostable Cas protein performs (e.g., its collateral cleavage activity functions sufficiently) within a temperature range at which nucleic acid extension and/or amplification reaction(s) are performed; those skilled in the art are well familiar with various such reactions and the temperature ranges at which they are performed, In some embodiments, such a temperature range may be above a temperature selected from the group consisting of about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., about 80° C., about 81° C., about 82° C., about 83° C., about 84° C., about 85° C., about 86° C., about 87° C., about 88° C., about 89° C., about 90° C., about 91° C., about 92° C., about 93° C., about 94° C., about 95° C., about 96° C., about 97° C., about 98° C., about 99° C., about 100° C., or combinations thereof. In some embodiments, a temperature range may be about 60° C. to about 90° C. In some embodiments, a temperature range may be about 60° C. to about 80° C. In some embodiments, a temperature range may be about 60° C. to about 75° C. In some embodiments, a temperature range may be about 65° C. to about 90° C. In some embodiments, a temperature range may be about 60° C. to about 80° C. In some embodiments, a temperature range may be about 60° C. to about 75° C.
Thus, as is set forth herein, in some embodiments, a useful thermostable Cas protein is a Cas12 or Cas13 homolog (e.g., ortholog), e.g., a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99% or 100% sequence identity to any one of SEQ ID Nos. 1-283 that is thermostable at temperatures above about 50° C., and in some embodiments above about 60° C., for example within and/or above about 60-65° C. Those skilled in the art, reading the present disclosure will particularly appreciate that, in some embodiments, a useful thermostable Cas protein is a Cas12 (e.g., SEQ ID NO 3-21, 33-47, 51-56, 68-178, and 274-283, or a variant thereof, for example having at least 90%, 95%, 99% or greater amino acid sequence identity thereto) or Cas13 (e.g., SEQ ID NO 1-2, 22-32, 48-50, 57-67, 179-273, or a variant thereof, for example having at least 90%, 95%, 99% or greater amino acid sequence identity thereto) whose activity (e.g., whose target binding and collateral cleavage activities) is sufficiently thermostable, for example at temperatures within a range of 60-65° C. to perform in assays as described herein (e.g., in some embodiments, one-pot assays). For example, in some embodiments, sufficient thermostable activity is activity that is reasonably comparable to (e.g., within about 25%) of an appropriate reference thermostable Cas protein (e.g., SEQ ID NO 15) as described herein.
In some embodiments, the disclosure describes a detection method comprising steps of: contacting a CRISPR-Cas complex comprising: a Cas protein with collateral cleavage activity that is thermostable at temperatures above at least 60-65° C.; and a guide RNA selected or engineered to be complementary to a target sequence; with a sample potentially comprising a nucleic acid of the target sequence.
In some embodiments, the step of contacting comprises contacting the CRISRP-Cas complex and sample with a reporter susceptible to cleavage by the Cas protein collateral activity. In some embodiments, the step of contacting comprises incubating for a period of time above the temperature. In some embodiments, a detection method further comprises a step of amplifying nucleic acid present in the sample. In some embodiments, the step of amplifying utilizes a thermostable nucleic acid polymerase. In some embodiments, the steps of amplifying and contacting are performed in a single vessel.
In some embodiments, the Cas protein is a Cas12 protein. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15. In some embodiments, the Cas protein has an amino acid sequence having at least 80%, sequence identity to any one of SEQ ID Nos. 3-21, 33-47, 51-56, 68-178, and 274-283. In some embodiments, the Cas protein has an amino acid sequence having 80%, sequence identity to any one of SEQ ID Nos. 1-283.
In some embodiments, in a method of performing a detection assay utilizing a Cas protein with collateral cleavage activity, the improvement that comprises utilizing a Cas protein with thermostable collateral cleavage activity. In some embodiments, the Cas protein is a Cas12 protein. In some embodiments, the Cas protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15. In some embodiments, the Cas protein has an amino acid sequence having at least 80%, sequence identity to any one of SEQ ID Nos. 3-21, 33-47, 51-56, 68-178, and 274-283. In some embodiments, a method of performing a detection assay is conducted in a single reaction vessel. In some embodiments, the thermostable collateral cleavage activity is thermostable above a temperature of about 60° C. In some embodiments, the thermostable collateral cleavage activity is thermostable above a temperature of about 65° C. In some embodiments, the Cas protein has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID Nos. 1-283.
Those skilled in the art are well aware of the burgeoning plethora of useful detection (e.g., diagnostic) assays that have been and are being developed using Cas protein collateral activities. See, for example, Sashital Genome Med 2018:10, 32. Furthermore, those skilled in the art are well aware that a “detailed classification of CRISPR/Cas biosensing systems” based on Cas protein collateral activity has recently been made publicly available. See review by Li et al Trends Biotechnol. 37:730, July 2019.
Formats of particular interest include Cas13-based (e.g., Cas13a- or Cas13b-based) systems, including those referenced as “SHERLOCK” and/or “HUDSON” systems (see, for example, Gootenberg et al, Science 356:438, 2017; Gootenberg et al, Science 360:339, 2018; Myhrvold et al., Science 360:444, 2018; see also U.S. Ser. No. 10/266,887) and Cas12-based (e.g., Cas12a- or Cas12b-based) systems, including those references as “HOLMES” or “DETECTR” systems (see, for example, Cheng et al. CN patent filing CN107488710A; PCT/CN18/82769 and U.S. Ser. No. 16/631,157; Li et al. Cell Disc. 4:20, 2018; Chen et al. Science 360:436, 2018; Li, L. et al. bioRxiv Published online Jul. 26, 2018. http://dx.doi.org/10.1101/362889; U.S. Ser. No. 10/253,365). Both Cas13a and Cas13b enzymes have been used in SHERLOCK and/or HUDSON systems; similarly both Cas12a and Cas12b.
As is known in the art, and described in references cited herein, typical detection assays that utilize Cas protein collateral cleavage activity involve contacting an appropriate CRISPR-Cas complex, including a Cas protein with collateral activity and a guide RNA complementary to a target sequence of interest, with a sample that may contain the target sequence. Upon recognition of the target sequence, the Cas protein's collateral activity is activated, so that it cleaves unrelated nucleic acid (DNA or RNA or both, depending on the enzyme). A reporter of the relevant cleavable nucleic acid is provided, appropriately configured (e.g., labeled) so that its cleavage as a result of the activated collateral activity is detectable (e.g., separates a fluorophore from a quencher so that fluorescence becomes detectable, etc).
In many assays, a target sequence is generated and/or amplified (e.g., copied from RNA to DNA and/or amplified, for example by primer extension, DNA replication (e.g., by polymerase chain reaction) and/or transcription). See, for example,
Thus, in many embodiments, a collateral activity assay includes steps of (1) target copying and/or amplification; (2) target binding; and (3) signal release and/or detection.
Typically, collateral activity assays as described herein are in vitro assays. In some embodiments, they may be cell free assays (e.g., may be substantially free of intact cells, or, in some embodiments, of cell fragments).
In some embodiments, collateral activity assays as described herein are performed on samples that are or are prepared from biological (e.g., blood, saliva, tears, urine, etc) or environmental (e.g., soil, water, etc) primary samples.
Thermostable Cas Enzymes
As described herein, the present disclosure identifies the source of a problem with certain detection (e.g., diagnostic assays) that utilize Cas protein collateral activity, as described above, in that certain Cas proteins with collateral activity are insufficiently stable at relevant temperatures (e.g., at temperatures at which nucleic acid extension and/or amplification are performed). Additionally, The present disclosure further surprisingly demonstrates that, for some proteins, loss of activity upon temperature elevation may be irreversible. This reality increases the significance of the insight, provided by the present disclosure, that Cas proteins with thermostable collateral activity are particularly desirable for use in assays asia described herein.
The present disclosure therefore provides improved detection (e.g., diagnostic) assays that utilize Cas protein collateral activity, which improved assays utilize a thermostable Cas protein (e.g., whose collateral activity is thermostable) as described herein.
In some embodiments, steps of nucleic acid detection and target binding are performed in a single vessel; in some embodiments, steps of target binding an signal release are performed in a single vessel; in some embodiments, steps of steps of (1) target copying and/or amplification; (2) target binding; and (3) signal release and/or detection are performed in a single vessel; in some embodiments all steps are performed in a single vessel—i.e., provided improved assays are one-pot assays.
In some embodiments, improved collateral activity assays as described herein are in vitro assays. In some embodiments, they may be cell free assays (e.g., may be substantially free of intact cells, or, in some embodiments, of cell fragments).
In some embodiments, improved collateral activity assays as described herein are performed on samples that are or are prepared from biological (e.g., blood, saliva, tears, urine, etc) or environmental (e.g., soil, water, etc) primary sample.
In some embodiments, a Cas enzyme with thermostable collateral cleavage activity is a homolog (e.g., ortholog) of a Cas enzyme that either does not have demonstrable collateral cleavage activity, or has demonstrable collateral cleavage activity but loses such activity above a relevant temperature as described herein.
In some embodiments, a Cas enzyme with thermostable collateral cleavage activity as described herein is a Cas12 (e.g., Cas12a or Cas12b) enzyme. In some embodiments, a Cas enzyme with thermostable collateral cleavage activity as described herein is a Cas13 (e.g., Cas13a or Cas13b) enzyme.
In some embodiments, a Cas enzyme with thermostable collateral cleavage activity as described herein is a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99% or 100% sequence identity to any one of SEQ ID Nos. 1-283. In some embodiments, improved collateral activity assays as described herein are performed using a Cas enzyme comprising an amino acid sequence having 80%, 85%, 90%, 99% or 100% sequence identity to any one of SEQ ID Nos. 1-283.
Target Nucleic Acid
Those skilled in the art will immediately appreciate that technologies provided herein are broadly applicable to achieve detection of a wide range of nucleic acids including, for example, nucleic acids from an infectious agent (e.g., a virus, microbe, parasite, etc), nucleic acids indicative of a particular physiological state or condition (e.g., presence or state of a disease, disorder or condition such as, for example, cancer or an inflammatory or metabolic disease, disorder or condition, etc), prenatal nucleic acids, etc.
In some embodiments, a target nucleic acid is detected by an assay comprising a Cas enzyme as described herein and a cRNA. In some embodiments, the structure of the cRNA can affect the activity of the Cas/cRNA complex. In some embodiments the structure of the Cas/cRNA complex contributes to the thermostability of the Cas collateral activity.
Typically, provided technologies will be applied to one or more samples to assess presence and/or level of one or more target nucleic acids in the sample. In some embodiments, the sample is a biological sample; in some embodiments, a sample is an environmental sample. In some embodiments, a sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing).
In some embodiments, a sample will be processed (e.g., nucleic acids will be partially or substantially isolated or purified out of a primary sample); in some embodiments, only minimal processing will have been performed (i.e., the sample will be a crude sample).
The thermostability of LwaCas13a was tested. Briefly, labeled RNA target was incubated with Rnase Inhibitor; T7 RNA Polymerase, LwaCas13a, MgCl2 and a cRNA. Individual samples were incubated at various temperatures to determine collateral activity.
Further,
The thermostability of AsCas12a and Lbacas12a was tested. Briefly, labeled RNA target was incubated with Rnase Inhibitor; T7 RNA Polymerase, AsCas12a or Lbacas12a, MgCl2 and a cRNA. Individual samples were incubated at various temperatures to determine collateral activity.
The present Example describes certain thermostable Cas13 candidates for use in improved collateral activity assays as described herein.
In the present Example, it was determined that a Cas13 with collateral activity thermostable within a temperature range of about 62-about 68° C. would be particularly desirable, among other things, in one-pot assays with LAMP pre-amplification.
We performed a computational search for potentially thermostable Cas candidates and identified:
Exemplary sequences for use with TccCas13a include, but are not limited to:
Exemplary sequences for use with ThpCas13a include, but are not limited to:
Exemplary sequences for use with AacCas12b include, but are not limited to:
Exemplary sequences for use with AkCas12b include, but are not limited to:
Exemplary sequences for use with BhCas12b include, but are not limited to:
Exemplary sequences for use with LsCas12b include, but are not limited to:
Exemplary sequences of Cas proteins with thermostable collateral activity include those described in Table 1:
Table 1: Exemplary Sequences of Cas Proteins with Thermostable Collateral Activity
caenicola)
profundimaris)
acidoterrestris)
kakegawensis)
hisashii)
sediminis)
Those skilled in the art will appreciate that, given that their amino acid sequences are known, these enzymes can readily be produced (e.g., through culturing of source organisms and/or by recombinant expression/purification, as, for example, may be contracted from any of a variety of commercial sources). Produced enzymes can then be assessed for direct and/or collateral cleavage at varying temperature(s) and/or for other evidence of stability and/or functionality at relevant temperature(s).
This example confirms and further demonstrates the thermostability of Cas13 enzymes. This example provides certain thermostable Cas13 candidates for use in improved collateral activity assays as described herein. The thermostability of TccCas13a and ThpCas13a was tested. Briefly, varying ranges of labeled RNA target was incubated with TccCas13a or ThpCas13a; Rnase Inhibitor; T7 RNA Polymerase, MgCl2 and a cRNA (either in forward or reverse complement orientation).
Additional assays were performed with TccCas13a with or without some components to identify parameters contributing to background within the assays.
The present example demonstrates an exemplary method of discovery and screening thermostable Cas enzyme candidates (e.g., Cas12 and Cas13 enzymes) (
Candidate enzymes were expressed by in vitro protein synthesis (e.g. PURExpress in vitro Protein Synthesis Kit by New England BioLabs) according to manufacturer's instructions. An initial pool of Cas12a candidate enzymes were assessed by endpoint (
A subset of candidates (e.g., 12 candidates) that demonstrated highest activity among the 44 initial candidates at 52° C. were selected in combination with their most efficient guide and target for further assessment at higher temperatures (e.g., 58° C., 60° C., 62° C.).
Both endpoint and kinetic analyses indicated a subset of candidate enzymes (e.g., 9 of 12 candidate enzymes) that displayed some activity at 58° C. Of the nine with some activity, five were classified as high activity (RS9, RS12, RS38, RS54, and RS56), and the remaining four were classified as lower activity (RS31, RS39, RS47 and RS50) (
Both end point and kinetic analyses indicated a subset (e.g., 5 of 12 candidate enzymes) that displayed some activity at 60° C. Of the five with some activity, three were classified as high activity (RS50, RS56, and RS9), and the remaining two were classified as lower activity (RS28 and RS29) (
Both end point and kinetic analyses indicated a subset (e.g., 2 of 12 candidate enzymes) that displayed some activity at 62° C. (RS9 and RS54) (
While the refined list of 12 candidate enzymes were assessed at varying temperature, four priority candidate enzymes (RS10, RS28, RS38, and RS54) were purified and assessed for activity at varying temperatures (e.g., approximately 35° C. to approximately 65° C.). RS54 showed activity at LAMP temperatures (e.g., 61° C.) (
A subset of Cas12a candidates were further examined using three different guide and target sets compared to no template control at both 58° C. and 70° C. (
An initial pool of Cas13 candidate enzymes were assessed by endpoint analysis (
The present example demonstrates characterization of an exemplary thermostable Cas12a enzyme, RS9. To determine whether RS9 required use of Thermostable Inorganic Pyrophosphatase (TIPP) for amplification of a target nucleic acid, real time detection of ORF1ab amplification was completed over a range of starting concentrations of ORF1ab (4.5 copies/4 to 4,500 copies/4) in the presence or absence of TIPP. An exemplary reaction included 30 ng/ul RS9, 112.5 XL-213 (ORF1ab guide), 1× HKFB (ORF1ab) primer set, 1× wsLAMP mix, 125 nM DNase Alert, with or without 1 U Thermostable Inorganic Pyrophosphatase (TIPP) with indicated viral RNA template concentration present. The exemplary reaction was incubated at 58° C. for 120 minutes on QS5 with detection in VIC channel. Real-time reactions that did not contain TIPP resulted in no statistically significant amplification of ORF1ab compared to the no template control regardless of the starting concentration of template. Real-time reactions that did contain TIPP, with the exception of that with a starting concentration of template at 4.5 copies/4, demonstrated significant amplification of ORF1ab compared to the no template control, suggesting RS9 requires use of TIPP for amplification (
Specificity of RS9 was also assessed using real-time analysis. Amplification was conducted with a starting concentration of 4,500 copies/μL ORF1ab template and either primers and guides specific to ORF1ab or non-targeting primers with an ORF1ab guide. Each reaction condition was also conducted in the presence or absence of TIPP. An exemplary reaction included 30 ng/ul RS9, 112.5 XL-213 (ORF1ab guide), 1×HKFB (ORF1ab) or CFB (N) primer set, 1× wsLAMP mix, 125 nM DNase Alert, with or without 1 U Thermostable Inorganic Pyrophosphatase (TIPP), with 4,500 copies/μl viral RNA present. An exemplary reaction was incubated at 58° C. for 120 minutes on QS5 and detected in VIC channel. No amplification was detected for reactions containing non-targeting primers and/or no TIPP. Robust amplification was detected in reactions containing ORF1ab primers, an ORF1ab guide, and TIPP, indicating RS9 is specific for its target and requires TIPP for amplification (
RS9 collateral cleavage activity was assessed using 100 nM of single stranded DNA target and DNaseAlert or RNaseAlert as reporters. A no target condition was utilized as a negative control. RS9 was unable to cleave DNaseAlert, resulting in intensity measurements significantly above no target control conditions. RS9 was able to cleave RNaseAlert, resulting in measured intensity similar to that of the no target control conditions, indicating RS9 has RNA-specific collateral cleavage activity (
RS9 collateral cleavage activity was further evaluated alongside known Cas12a, LbaCas12a, using ORF LAMP product as target and either RNaseAlert, PolyrA, PolyrC, or PolyrU reporters. A no target condition was utilized as a negative control. Both RS9 and LbaCas12a were able to cleave RNaseAlert more efficiently than either PolyrA, PolyrC, or PolyrU (
The present example demonstrates characterization of an exemplary thermostable Cas13a enzyme, TccCas13a. To determine optimal temperature for TccCas13a activity, a Cas reaction was conducted over a range of temperatures using 10 nM target and RNaseAlert as reporter. A no target condition was utilized as a negative control. Temperature profiles suggest TccCas13a shows highest activity at approximately 62° C. (
To determine whether TccCas13a could be activated by ssDNA in addition to RNA, a Cas reaction was completed at 62° C. with RNaseAlert as a reporter. Different targets were utilized at different concentrations (e.g., 10 nM, 100 nM, or 1,000 nM ssDNA or 10 nM RNA). A no target condition was utilized as a negative control. Results indicated that TccCas13a can be activated by RNA, but cannot be activated by ssDNA even at the highest concentrations of ssDNA template at 62° C. (
TccCas13a activation by ssDNA was also assessed at 58° C. TccCas13a was activated at 58° C. in the presence of 1 nM, 10 nM, and 100 nM RNA target compared to no target control, while TccCas13a activation by ssDNA at 58° C. was only detected when 100 nM or 1,000 nM of ssDNA target was utilized. 10 nM of ssDNA target at 58° C. showed no difference compared to no target control, suggesting TccCas13a activation requires a higher concentration of ssDNA than RNA, similar to that observed for LwaCas13 ssDNA activation. Interestingly, TccCas13a was not activated by ssDNA* at any concentration (10 nM, 100 nM, or 1,000 nM) compared to control (no target) (
To determine whether TccCas13a showed different specific collateral activity to that of LwaCas13, a Cas reaction was done with two different reporters, RNaseAlert which contains multiple different bases (“NN”) and a “UU”-specific reporter with only two “UU” bases and a DNA backbone. Reactions were conducted at 60° C. LwaCas13a showed no preference for collateral activity of either reporter over the other, while TccCas13a showed increased collateral activity at “NN” sites compared to “UU” sites (
The present example demonstrates characterization of exemplary thermostable Cas enzymes, Pal1 (SEQ ID NO. 274), Pal2 low MW, Pal2 high MW (SEQ ID NO. 275), and Pal3 (SEQ ID NO. 276). Each enzyme was tested with four guides (designated as 342-353) at both 37° C. and 56° C. in a Cas-only reaction with DnaseAlert as a reporter. Fluorescence signal was plotted vs. time for each reaction (
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:
This application claims priority to each of U.S. Provisional Patent Application Nos. 62/966,527; filed Jan. 27, 2020; 62/967,536; filed Jan. 29, 2020; 62/970,159; filed Feb. 4, 2020; 63/038,710; filed Jun. 12, 2020; 63/139,267; filed Jan. 19, 2021 the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/015306 | 1/27/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63139267 | Jan 2021 | US | |
| 63038710 | Jun 2020 | US | |
| 62970159 | Feb 2020 | US | |
| 62967536 | Jan 2020 | US | |
| 62966527 | Jan 2020 | US |
